Engineering Of Cell Factories Research Articles

Application of multiple strategies to enhance oleanolic acid biosynthesis by engineered Saccharomyces cerevisiae

Oleanolic acid and its derivatives are widely used in the pharmaceutical, agricultural, cosmetic and food industries. Previous studies have shown that oleanolic acid production levels in engineered cell factories are low, which is why oleanolic acid is still widely extracted from traditional medicinal plants. To construct a highly efficient oleanolic acid production strain, rate-limiting steps were regulated by inducible promoters and the expression of key genes in the oleanolic acid synthetic pathway was enhanced. Subsequently, precursor pool expansion, pathway refactoring and diploid construction were considered to harmonize cell growth and oleanolic acid production. The multi-strategy combination promoted oleanolic acid production of up to 4.07 g/L in a 100 L bioreactor, which was the highest level reported.

Proteomic analysis reveals that the co-ordination of cytosolic and mitochondrial pathways is beneficial for sabinene biosynthesis in engineered Saccharomyces cerevisiae.

Reconstruction and optimization of biosynthetic pathways can help to overproduce target chemicals in microbial cell factories based on genetic engineering. However, the perturbation of biosynthetic pathways on cellular metabolism is not well investigated and profiling the engineered microbes remains challenging. The rapid development of omics tools has the potential to characterize the engineered microbial cell factory. Here, we performed label-free quantitative proteomic analysis and metabolomic analysis of engineered sabinene overproducing Saccharomyces cerevisiae strains. Combined metabolic analysis andproteomic analysis of targeted mevalonate (MVA) pathway showed that co-ordination of cytosolic and mitochondrial pathways had balanced metabolism, and genome integration of biosynthetic genes had higher sabinene production with less MVA enzymes. Furthermore, comparative proteomic analysis showed that compartmentalized mitochondria pathway had perturbation on central cellular metabolism. This study provided an omics analysis example for characterizing engineered cell factory, which can guide future regulation of the cellular metabolism and maintaining optimal protein expression levels for the synthesis of target products.

Dual cytoplasmic-peroxisomal compartmentalization engineering and multiple metabolic engineering strategies for high yield non-psychoactive cannabinoid in Saccharomyces cerevisiae.

CBG (Cannabigerol), a nonpsychoactive cannabinoid, has garnered attention due to its extensive antimicrobial and anti-inflammatory properties. However, the natural content of CBG in Cannabis sativa L. is minimal. In this study, we developed an engineered cell factory for CBG production using Saccharomyces cerevisiae. We introduced the CBGA biosynthetic pathway into S. cerevisiae and employed several strategies to enhance CBGA production. These strategies included dynamically inhibiting the competitive bypass of key metabolic pathways regulated by Erg20p. Additionally, we implemented a dual cytoplasmic-peroxisomal compartmentalization approach to further increase CBGA production. Furthermore, we ensured efficient CBGA production by optimizing NADPH and acetyl-CoA pools. Ultimately, our engineered strain achieved a CBG titer of 138mgL-1 through fed-batch fermentation in a 5 L bioreactor, facilitated by microwave decarboxylation extraction. These findings underscore the significant potential of yeast cell factories for achieving higher yields in cannabinoid production.

Novel cytochrome P450s for various hydroxylation of steroids from filamentous fungi

Hydroxylated steroids are value-added products with diverse biological activities mediated by cytochrome P450 enzymes, however, few has been thoroughly characterized in fungi. This study introduces a rapid identification strategy for filamentous fungi P450 enzymes through transcriptome and bioinformatics analysis. Five novel enzymes (CYP68J5, CYP68L10, CYP68J3, CYP68N1 and CYP68N3) were identified and characterized in Saccharomyces cerevisiae or Aspergillus oryzae. Molecular docking and dynamics simulations were employed to elucidate hydroxylation preferences of CYP68J5 (11α, 7α bihydroxylase) and CYP68N1 (11α hydroxylase). Additionally, redox partners (cytochrome P450 reductase and cytochrome b5) and ABC transporter were co-expressed with CYP68N1 to enhance 11α-OH-androstenedione (11α-OH-4AD) production. The engineered cell factory, co-expressing CPR1 and CYP68N1, achieved a significant increase of 11α-OH-4AD production, reaching 0.845 g·L-1, which increased by 14 times compared to the original strain. This study provides a comprehensive approach for identifying and implementing novel cytochrome P450 enzymes, paving the way for sustainable production of steroidal products.

Intensification and techno-economic analysis of a biocatalytic process for coniferol production from ferulic acid: An innovative strategy for lignin valorization

Coniferol, as a natural monolignol, acts as a chemical building block for various high-value compounds. However, the current petrochemical synthesis of coniferol and its limited scalability raise environmental and cost concerns. A biocatalytic method for coniferol production from its natural precursor, ferulic acid, was previously developed using an engineered cell factory that co-expresses carboxylic acid reductase and aldo-keto reductase. The present study focused on investigating the limiting factors of coniferol production and optimizing the process using the design of experiments and fed-batch approaches. By introducing novel parameters, including an inoculum of 0.25 g/L cells and a starting concentration of 5.0 mM ferulic acid, followed by two fed-batch additions of 2.0 mM ferulic acid and 3.7 g/L glycerol, coniferol production was enhanced by a factor of 2 (reaching 1.6 g/L). The scale-up process in a 1 L bioreactor and its intensified technical and economic analysis demonstrated its cost-effectiveness. Considering two different purchase prices for the ferulic acid of $5.0 and $650.0 per kg, the payback time was estimated at 3.3 and 3.2 years, respectively, and the minimal selling price of coniferol to obtain a IRR of 30% was calculated as $4.2 and $5.0 per gram, which is significantly lower than the current market price. In conclusion, the findings illustrate a biocatalytic approach for producing valuable compounds with potential applications in various industries, including cosmetics and pharmaceuticals. Furthermore, this approach contributes to developing sustainable and profitable biorefinery processes, paving the way for future initiatives focused on biocatalytic lignin valorization.

Green Manufacturing of Steroids via Mycolicbacteria: Current Status and Development Trends

Steroids, the second largest drug category ranked after antibiotics, find widespread use in treatments for reproductive health, endocrine regulation, and inflammation. Advances in steroidal chemistry to date have led to the widespread use of sterols as starting substances in the development of environmentally friendly biotechnologies for steroid production, including biocatalysis, microbial transformations, and biosynthesis using engineered micro-organisms. In this review, we synthesize some of the recent advancements in steroid biocatalysis using the Mycolicibacterium species, including the identification and modification of crucial elements for enhanced production. We also delve into the detailed characterization and reconstruction of metabolic pathways in specific microbial strains, shedding light on their potential for steroid biosynthesis. Additionally, we highlight the development of innovative de novo biosynthesis pathways for steroids within engineered cell factories. These results collectively provide an overview of the current landscape and emerging trends in green steroid manufacturing within the steroidal pharmaceutical industry.

Optimal energy and redox metabolism in the cyanobacterium Synechocystis sp. PCC 6803

Understanding energy and redox homeostasis and carbon partitioning is crucial for systems metabolic engineering of cell factories. Carbon metabolism alone cannot achieve maximal accumulation of metabolites in production hosts, since an efficient production of target molecules requires energy and redox balance, in addition to carbon flow. The interplay between cofactor regeneration and heterologous production in photosynthetic microorganisms is not fully explored. To investigate the optimality of energy and redox metabolism, while overproducing alkenes—isobutene, isoprene, ethylene and 1-undecene, in the cyanobacterium Synechocystis sp. PCC 6803, we applied stoichiometric metabolic modelling. Our network-wide analysis indicates that the rate of NAD(P)H regeneration, rather than of ATP, controls ATP/NADPH ratio, and thereby bioproduction. The simulation also implies that energy and redox balance is interconnected with carbon and nitrogen metabolism. Furthermore, we show that an auxiliary pathway, composed of serine, one-carbon and glycine metabolism, supports cellular redox homeostasis and ATP cycling. The study revealed non-intuitive metabolic pathways required to enhance alkene production, which are mainly driven by a few key reactions carrying a high flux. We envision that the presented comparative in-silico metabolic analysis will guide the rational design of Synechocystis as a photobiological production platform of target chemicals.

Membrane manipulation by free fatty acids improves microbial plant polyphenol synthesis

Microbial synthesis of nutraceutically and pharmaceutically interesting plant polyphenols represents a more environmentally friendly alternative to chemical synthesis or plant extraction. However, most polyphenols are cytotoxic for microorganisms as they are believed to negatively affect cell integrity and transport processes. To increase the production performance of engineered cell factories, strategies have to be developed to mitigate these detrimental effects. Here, we examine the accumulation of the stilbenoid resveratrol in the cell membrane and cell wall during its production using Corynebacterium glutamicum and uncover the membrane rigidifying effect of this stilbenoid experimentally and with molecular dynamics simulations. A screen of free fatty acid supplements identifies palmitelaidic acid and linoleic acid as suitable additives to attenuate resveratrol’s cytotoxic effects resulting in a three-fold higher product titer. This cost-effective approach to counteract membrane-damaging effects of product accumulation is transferable to the microbial production of other polyphenols and may represent an engineering target for other membrane-active bioproducts.

Improved Membrane Permeability via Hypervesiculation for In Situ Recovery of Lycopene in Escherichia coli.

Lycopene biosynthesis is frequently hampered by downstream processing hugely due to its inability to be secreted out from the producing chassis. Engineering cell factories can resolve this issue by secreting this hydrophobic compound. A highly permeable E.coli strain was developed for a better release rate of lycopene. Specifically, the heterologous mevalonate pathway and crtEBI genes from Corynebacterium glutamicum were overexpressed in Escherichia coli BL21 (DE3) for lycopene synthesis. To ensure in situ lycopene production, murein lipoprotein, lipoprotein NlpI, inner membrane permease protein, and membrane-anchored protein in TolA-TolQ-TolR were deleted for improved membrane permeability. The final strain, LYC-8, produced 438.44 ± 8.11 and 136.94 ± 1.94 mg/L of extracellular and intracellular lycopene in fed-batch fermentation. Both proteomics and lipidomics analyses of secreted outer membrane vesicles were perfect indicators of hypervesiculation. Changes in the ratio of saturated fatty acids, unsaturated fatty acids, and cyclopropane fatty acids coupled with the branching and acyl chain lengths altered the membrane fatty acid composition. This ensured membrane fluidity and permeability for in situ lycopene release. The combinatorial deletion of these genes altered the cellular morphology. The structural and morphological changes in cell shape, size, and length were associated with changes in the mechanical strength of the cell envelope. The enhanced lycopene production and secretion mediated by improved membrane permeability established a cell lysis-free system for an efficient releasing rate and downstream processing, demonstrating the importance of vesicle-associated membrane permeability in efficient lycopene production.

Modular Metabolic Engineering of Mortierella alpina by the 2A Peptide Platform to Improve Arachidonic Acid Production.

Arachidonic acid (ARA) is an essential fatty acid in human nutrition. Mortierella alpina, a filamentous fungus, has been widely used for the production of ARA. Here, we report a modular engineering approach that systematically eliminates metabolic bottlenecks in the multigene elongase/desaturase pathway and has led to significant improvements of the ARA titer. The elongase/desaturase pathway in Mortierella alpina was recast into two modules, namely, push and pull modules, based on its function in the ARA synthesis. Combinatorial optimization of these two modules has balanced the production and consumption of intermediate metabolites. A 2A peptide-based facile assembly platform that can achieve multigene expression as a polycistron was first established. The platform was then applied to express the push and pull modules in Mortierella alpina. In the shake-flask fermentation, the lipid and ARA contents of the engineered strain MA5 were increased by 1.2-fold and 77.6%, respectively, resulting in about fivefold increase of the ARA yield. The final ARA titer reached 4.4 g L-1 in shake-flask fermentation. The modular engineering strategies presented in this study demonstrate a generalized approach for the engineering of cell factories in the production of valuable metabolites.

An Overview of Sugar Nucleotide-Dependent Glycosyltransferases for Human Milk Oligosaccharide Synthesis.

Human milk oligosaccharides (HMOs) have received increasing attention because of their special effects on infant health and commercial value as the new generation of core components in infant formula. Currently, large-scale production of HMOs is generally based on microbial synthesis using metabolically engineered cell factories. Introduction of the specific glycosyltransferases is essential for the construction of HMO-producing engineered strains in which the HMO-producing glycosyltransferases are generally sugar nucleotide-dependent. Four types of glycosyltransferases have been used for typical glycosylation reactions to synthesize HMOs. Soluble expression, substrate specificity, and regioselectivity are common concerns of these glycosyltransferases in practical applications. Screening of specific glycosyltransferases is an important research topic to solve these problems. Molecular modification has also been performed to enhance the catalytic activity of various HMO-producing glycosyltransferases and to improve the substrate specificity and regioselectivity. In this article, various sugar nucleotide-dependent glycosyltransferases for HMO synthesis were overviewed, common concerns of these glycosyltransferases were described, and the future perspectives of glycosyltransferase-related studies were provided.

Nanodroplet-Based Reagent Delivery into Water-in-Fluorinated-Oil Droplets.

In vitro compartmentalization (IVC) is a technique for generating water-in-oil microdroplets to establish the genotype (DNA information)-phenotype (biomolecule function) linkage required by many biological applications. Recently, fluorinated oils have become more widely used for making microdroplets due to their better biocompatibility. However, it is difficult to perform multi-step reactions requiring the addition of reagents in water-in-fluorinated-oil microdroplets. On-chip droplet manipulation is usually used for such purposes, but it may encounter some technical issues such as low throughput or time delay of reagent delivery into different microdroplets. Hence, to overcome the above issues, we demonstrated a nanodroplet-based approach for the delivery of copper ions and middle-sized peptide molecules (human p53 peptide, 2 kDa). We confirmed the ion delivery by microscopic inspection of crystal formation inside the microdroplet, and confirmed the peptide delivery using a fluorescent immunosensor. We believe that this nanodroplet-based delivery method is a promising approach to achieving precise control for a broad range of fluorocarbon IVC-based biological applications, including molecular evolution, cell factory engineering, digital nucleic acid detection, or drug screening.

Remodeling Isoprene Pyrophosphate Metabolism for Promoting Terpenoids Bioproduction

Terpenoids are the largest family of natural products. They are made from the building block isoprene pyrophosphate (IPP), and their bioproduction using engineered cell factories has received a great deal of attention. To date, the insufficient metabolic supply of IPP remains a great challenge for the efficient synthesis of terpenoids. In this work, we discover that the imbalanced metabolic flux distribution between the central metabolism and the IPP supply hinders IPP accumulation in Bacillus subtilis (B. subtilis). Therefore, we remodel the IPP metabolism using a series of genetically encoded two-input-multi-output (TIMO) circuits that are responsive to pyruvate or/and malonyl-CoA, resulting in an IPP pool that is significantly increased by up to four-fold. As a proof-of-concept validation, we design an IPP metabolism remodeling strategy to improve the production of three valuable terpenoids, including menaquinone-7 (MK-7, 4.1-fold), lycopene (9-fold), and β-carotene (0.9-fold). In particular, the titer of MK-7 in a 50-L bioreactor reached 1549.6 mg∙L−1, representing the highest titer reported so far. Thus, we propose a TIMO genetic circuits-assisted IPP metabolism remodeling framework that can be generally used for the synergistic fine-tuning of complicated metabolic modules to achieve the efficient bioproduction of terpenoids.

An improved integrative GFP-based vector for genetic engineering of Parageobacillus thermoglucosidasius facilitates the identification of a key sporulation regulator

Parageobacillus thermoglucosidasius is a thermophilic Gram-positive bacterium, which is a promising host organism for sustainable bio-based production processes. However, to take full advantage of the potential of P. thermoglucosidasius, more efficient tools for genetic engineering are required. The present study describes an improved shuttle vector, which speeds up recombination-based genomic modification by incorporating a thermostable sfGFP variant into the vector backbone. This additional selection marker allows for easier identification of recombinants, thereby removing the need for several culturing steps. The novel GFP-based shuttle is therefore capable of facilitating faster metabolic engineering of P. thermoglucosidasius through genomic deletion, integration, or exchange. To demonstrate the efficiency of the new system, the GFP-based vector was utilised for deletion of the spo0A gene in P. thermoglucosidasius DSM2542. This gene is known to be a key regulator of sporulation in Bacillus subtilis, and it was therefore hypothesised that the deletion of spo0A in P. thermoglucosiadius would produce an analogous sporulation-inhibited phenotype. Subsequent analyses of cell morphology and culture heat resistance suggests that the P. thermoglucosidasius ∆spo0A strain is sporulation-deficient. This strain may be an excellent starting point for future cell factory engineering of P. thermoglucosidasius, as the formation of endospores is normally not a desired trait in large-scale production.

Cell factory engineering: Challenges and opportunities for synthetic biology applications.

The production of high-quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with the proteotoxic stress induced upon aberrant accumulation of off-pathway protein folding intermediates, which eventually culminate in the induction of apoptosis. In this review, we will discuss advances in cell engineering and their applications at different hierarchical levels of control of the expression of recombinant proteins, from transcription and translational to posttranslational modifications and subcellular trafficking. We also highlight challenges and unique opportunities to apply modern synthetic biology tools to the design of programmable cell factories for improved biomanufacturing of therapeutic proteins.

In Silico Prediction and Mining of Exporters for Secretory Bioproduction of Terpenoids in Saccharomyces cerevisiae.

Terpenoids are the largest class of natural products, and their bioproduction by engineered cell factories receives high attention. However, excessive intracellular accumulation is one of the bottlenecks that limit the further improvement of the yield of terpenoid products. Therefore, it is important to mine exporters to achieve the secretory production of terpenoids. This study proposed a framework for the in silico prediction and mining of terpenoid exporters in Saccharomyces cerevisiae. Through the process of "mining-docking-construction-validation", we found that Pdr5 of ATP-binding cassette (ABC) transporters and Osh3 of oxysterol-binding homology (Osh) proteins can promote squalene efflux. Squalene secretion of the strain overexpressing Pdr5 and Osh3 increased to 141.1 times that of the control strain. Besides squalene, ABC exporters also can promote the secretion of β-carotene and retinal. Molecular dynamics simulation results revealed that before exporter conformations transitioned to the "outward-open" states, the substrates might have bound to the tunnels and prepared for rapid efflux. Overall, this study provides a terpenoid exporter prediction and mining framework that may be generally used to identify exporters of other terpenoids.

CRISPRi-microfluidics screening enables genome-scale target identification for high-titer protein production and secretion

Genome-scale target identification promises to guide microbial cell factory engineering for higher-titer production of biomolecules such as recombinant proteins (r-protein), but challenges remain due to the need not only for comprehensive genotypic perturbation but also in conjunction with high-throughput phenotypic screening strategies. Here, we developed a CRISPRi-microfluidics screening platform to systematically identify crucial gene targets that can be engineered to enhance r-protein secretion in Corynebacterium glutamicum. We created a CRISPR interference (CRISPRi) library containing 46,549 single-guide RNAs, where we aimed to unbiasedly target all genes for repression. Meanwhile, we developed a highly efficient droplet-based microfluidics system integrating the FlAsH-tetracysteine assay that enables screening of millions of strains to identify potential knockdowns conducive to nanobody VHH secretion. Among our highest-ranking candidates are a slew of previously unknown targets involved in transmembrane transport, amino-acid metabolism and redox regulation. Guided by these findings, we eventually constructed a hyperproducer for multiple proteins via combinatorial engineering of redox-response transcription factors. As the near-universal applicability of CRISPRi technology and the FlAsH-based screening platform, this procedure might be expanded to include a varied variety of microbial species and recombinant proteins.

Study of the persistence and dynamics of recombinant mCherry-producing Yarrowia lipolytica strains in the mouse intestine using fluorescence imaging.

Yarrowia lipolytica is a dimorphic oleaginous non-conventional yeast widely used as a powerful host for expressing heterologous proteins, as well as a promising source of engineered cell factories for various applications. This microorganism has a documented use in Feed and Food and a GRAS (generally recognized as safe) status. Moreover, in vivo studies demonstrated a beneficial effect of this yeast on animal health. However, despite the focus on Y. lipolytica for the industrial manufacturing of heterologous proteins and for probiotic effects, its potential for oral delivery of recombinant therapeutic proteins has seldom been evaluated in mammals. As the first steps towards this aim, we engineered two Y. lipolytica strains, a dairy strain and a laboratory strain, to produce the model fluorescent protein mCherry. We demonstrated that both Y. lipolytica strains transiently persisted for at least 1 week after four daily oral administrations and they maintained the active expression of mCherry in the mouse intestine. We used confocal microscopy to image individual Y. lipolytica cells of freshly collected intestinal tissues. They were found essentially in the lumen and they were rarely in contact with epithelial cells while transiting through the ileum, caecum and colon of mice. Taken as a whole, our results have shown that fluorescent Y. lipolytica strains constitute novel tools to study the persistence and dynamics of orally administered yeasts which could be used in the future as oral delivery vectors for the secretion of active therapeutic proteins in the gut.

Hierarchical dynamic regulation of Saccharomyces cerevisiaefor enhanced lutein biosynthesis.

Lutein, as a carotenoid with strong antioxidant capacity and an important component of macular pigment in the retina, has wide applications in pharmaceutical, food, feed, and cosmetics industries. Besides extraction from plant and algae, microbial fermentation using engineered cell factories to produce lutein has emerged as a promising route. However, intra-pathway competition between the lycopene cyclases and the conflict between cell growth and production are two major challenges. In our previous study, de novo synthesis of lutein had been achieved in Saccharomyces cerevisiae by dividing the pathway into two stages (δ-carotene formation and conversion) using temperature as the input signal to realize sequential cyclation of lycopene. However, lutein production was limited to microgram level, which is still too low to meet industrial demand. In this study, a dual-signal hierarchical dynamic regulation system was developed and applied to divide lutein biosynthesis into three stages in response to glucose concentration and culture temperature. By placing the genes involved in δ-carotene formation under the glucose-responsive ADH2 promoter and genes involved in the conversion of δ-carotene to lutein under temperature-responsive GAL promoters, the growth-production conflict and intra-pathway competition were simultaneously resolved. Meanwhile, the rate-limiting lycopene ε-cyclation and carotene hydroxylation reactions were improved by screening for lycopene ε-cyclase with higher activity and fine tuning of the P450 enzymes and their redox partners. Finally, a lutein titer of 19.92 mg/L (4.53 mg/g DCW) was obtained in shake-flask cultures using the engineered yeast strain YLutein-3S-6, which is the highest lutein titer ever reported in heterologous production systems.

Metabolic Engineering and Adaptive Evolution for Efficient Production of l-Lactic Acid in Saccharomyces cerevisiae.

l-Lactic acid (LA) is a three-carbon hydroxycarboxylic acid with extensive applications in food, cosmetic, agricultural, pharmaceutical, and bioplastic industries. However, microbial LA production is limited by its intrinsic inefficiency of cellular metabolism. Here, pathway engineering was used to rewire the biosynthetic pathway for LA production in Saccharomyces cerevisiae by screening heterologous l-lactate dehydrogenase, reducing ethanol accumulation, and introducing a bacterial acetyl coenzyme A (acetyl-CoA) synthesis pathway. To improve its intrinsic efficiency of LA export, transporter engineering was conducted by screening the monocarboxylate transporters and then strengthening the capacity of LA export, leading to LA production up to 51.4 g/L. To further enhance its intrinsic efficiency of acid tolerance, adaptive evolution was adopted by cultivating yeast cells with a gradual increase in LA levels during 12 serial subcultures, resulting in a 17.5% increase in LA production to 60.4 g/L. Finally, the engineered strain S.c-NO.2-100 was able to produce 121.5 g/L LA, with a yield of up to 0.81 g/g in a 5-L batch bioreactor. The strategy described here provides a guide for developing efficient cell factories for the production of the other industrially useful organic acids. IMPORTANCE Saccharomyces cerevisiae is one of the most widely engineered cell factories for the production of organic acids. However, microbial production of l-lactic acid is limited by its intrinsic inefficiency of cellular metabolism in S. cerevisiae. Here, the transmission efficiency of the biosynthetic pathway was improved by pathway optimization to increase l-lactic acid production. Then, the synthetic ability for l-lactic acid was further enhanced by adaptive evolution to improve acid tolerance of S. cerevisiae. Based on these strategies, the final engineered S. cerevisiae strain achieved high efficiency of l-lactic acid production. These findings provide new insight into improving the intrinsic efficiency of cellular metabolism and will help to construct superior industrial yeast strains for high-level production of other organic acids.