Metabolic Engineering Strategies Research Articles

A comprehensive review of spermidine: Safety, health effects, absorption and metabolism, food materials evaluation, physical and chemical processing, and bioprocessing.

Spermidine, a natural autophagy inducer, has a variety of health effects, such as antitumor, antiaging, anti-inflammation, cardiovascular protection, and neuromodulation. It has been a hot topic in the field of food processing, and current research findings suggest that spermidine-rich foods may be used in intervention and prevention of age-related diseases. In this article, recent findings on the safety, health effects, absorption and metabolism of spermidine were reviewed, and advances in food processing, including the raw materials evaluation, physical and chemical processing, and biological processing of spermidine, were highlighted. In particular, the core metabolic pathways, key gene targets, and efficient metabolic engineering strategies involved in the biosynthesis of spermidine and its precursors were discussed. Moreover, limitations and future perspectives of spermidine research were proposed. The purpose of this review is to provide new insights on spermidine from its safety to its food processing, which will advance the commercial production and applications of spermidine-rich foods and nutraceuticals.

Advances in metabolic engineering of cyanobacteria for production of biofuels

Current trends in fossil fuel use, having a direct impact on the global carbon cycle, could potentially be mitigated by adopting the use of biofuels with sustainable features. While bioethanol is currently being used as a major biofuel, especially for transportation worldwide, it is hygroscopic and contains less energy. Therefore, the prospect of biofuels has now shifted to higher alcohols, alkanes, and other molecules that have similar properties to existing gasoline and diesel fuels. Microalgae and cyanobacteria are aquatic photosynthetic microorganisms that can convert CO2 and water into carbon-rich lipids, a substrate for biodiesel production, more efficiently than the agricultural oleaginous crops, making them a superior option to traditional oil-seed crops. Cyanobacteria are gram-negative photosynthetic bacteria that have instrumental roles in global biological carbon sequestration, oxygen evolution, and nitrogen fixation. Furthermore, cyanobacteria have established genetic backgrounds, flexibility in genetic manipulation, and tolerance to the introduction of foreign genes. Metabolic engineering strategies in these photosynthetic hosts have resulted in the conception of cell factories capable of one-step synthesis of biofuel molecules from CO2, light, and water. Two freshwater cyanobacteria species, Synechocystis sp. PCC 6803 (Synechocystis 6803) and Synechococcus elongatus sp. PCC 7492 (Synechococcus 7492), and marine Synechococcus sp. PCC 7002 (Synechococcus 7002) are among the highly studied cyanobacteria for the production of biofuels. Here, we reviewed the recent progress in metabolic engineering and synthetic biology approaches in cyanobacteria for the synthesis of various biofuel molecules, ranging from alcohols to biodiesels and terpenes or terpenoids.

Metabolic Engineering Strategies for Improved Lipid Production and Cellular Physiological Responses in Yeast Saccharomyces cerevisiae

Microbial lipids have been a hot topic in the field of metabolic engineering and synthetic biology due to their increased market and important applications in biofuels, oleochemicals, cosmetics, etc. This review first compares the popular hosts for lipid production and explains the four modules for lipid synthesis in yeast, including the fatty acid biosynthesis module, lipid accumulation module, lipid sequestration module, and fatty acid modification module. This is followed by a summary of metabolic engineering strategies that could be used for enhancing each module for lipid production. In addition, the efforts being invested in improving the production of value-added fatty acids in engineered yeast, such as cyclopropane fatty acid, ricinoleic acid, gamma linoleic acid, EPA, and DHA, are included. A discussion is further made on the potential relationships between lipid pathway engineering and consequential changes in cellular physiological properties, such as cell membrane integrity, intracellular reactive oxygen species level, and mitochondrial membrane potential. Finally, with the rapid development of synthetic biology tools, such as CRISPR genome editing tools and machine learning models, this review proposes some future trends that could be employed to engineer yeast with enhanced intracellular lipid production while not compromising much of its cellular health.

Improving the Yield and Quality of Daptomycin in Streptomyces roseosporus by Multilevel Metabolic Engineering.

Daptomycin is a cyclic lipopeptide antibiotic with a significant antibacterial action against antibiotic-resistant Gram-positive bacteria. Despite numerous attempts to enhance daptomycin yield throughout the years, the production remains unsatisfactory. This study reports the application of multilevel metabolic engineering strategies in Streptomyces roseosporus to reconstruct high-quality daptomycin overproducing strain L2797-VHb, including precursor engineering (i.e., refactoring kynurenine pathway), regulatory pathway reconstruction (i.e., knocking out negative regulatory genes arpA and phaR), byproduct engineering (i.e., removing pigment), multicopy biosynthetic gene cluster (BGC), and fermentation process engineering (i.e., enhancing O2 supply). The daptomycin titer of L2797-VHb arrived at 113 mg/l with 565% higher comparing the starting strain L2790 (17 mg/l) in shake flasks and was further increased to 786 mg/l in 15 L fermenter. This multilevel metabolic engineering method not only effectively increases daptomycin production, but can also be applied to enhance antibiotic production in other industrial strains.

Crucial carotenogenic genes elevate hyperaccumulation of both fucoxanthin and β-carotene in Phaeodactylum tricornutum

Burgeoning demand for carotenoids from natural resources has urged to explore the promising metabolic engineering strategy to improve algal systems. However, existing biological intricacies in algal carotenogenesis warrant identification and exploitation of potential molecular candidate(s) for metabolic engineering to exploit the maximum potential of microalgal cell factories. Here, we identified two critical carotenogenic genes, including 1-deoxy-d-xylulose 5-phosphate synthase and lycopene cyclase and concurrently overexpressed in Phaeodactylum tricornutum. Molecular analysis proved the successful integration and expression of these two genes in engineered algal cells. Dual overexpression concurrently elevated fucoxanthin and β-carotene content up to 6.53 mg/g and 4.34 mg/g dry cell weight, respectively, which is the highest reported content of two carotenoids simultaneously, particularly the highest β-carotene content. Besides, dual overexpression did not impede algal physiological properties and biomass production. The findings exemplified the potential metabolic candidates capable of elevating the complex carotenogenesis and demonstrated a promising strategy to overcome the bottleneck in the commercial production of algal carotenoids.

Genetic regulation and fermentation strategy for squalene production in Schizochytrium sp.

Squalene, as an important terpenoid, is extensively used in the medicine and health care fields owing to its functions of anti-oxidation, blood lipid regulation and cancer prevention. The marine microalgae, Schizochytrium sp., which acts as an excellent strain with potential of high squalene production was selected as the starting strain. The overexpressed strain with sqs gene got the reduced biomass and lipid, while the squalene titer was increased by 79.6% ± 4.7% to 12.8 ± 0.2mg/L. In order to further increase squalene production, the recombinant strain (HS strain) with sqs and hmgr gene co-overexpression was further constructed. The biomass and squalene titer of the HS strain were increased by 13.6% ± 1.2% and 88.8% ± 5.3%, respectively, which indicated the carbon flux of the mevalonate pathway was enhanced for squalene accumulation. Regarding the squalene synthesis is completely coupled with cell growth, fermentation strategy to prolong the logarithmic growth phase was conducive to improve squalene production. Under the condition of optimal composition and concentrated medium, the squalene titer of HS strain was 27.0 ± 1.3mg/L, which was 2.0 times that of the basal medium condition (13.5 ± 0.4mg/L). This study which combined the metabolic engineering and fermentation strategy provides a new strategy for squalene production in Schizochytrium sp. KEY POINTS: •The overexpression of sqs and hmgr genes promoted carbon metabolism for squalene. •The optimal and concentrated media can increase squalene yield.

Biotechnological production of specialty aromatic and aromatic-derivative compounds.

Aromatic compounds are an important class of chemicals with different industrial applications. They are usually produced by chemical synthesis from petroleum-derived feedstocks, such as toluene, xylene and benzene. However, we are now facing threats from the excessive use of fossil fuels causing environmental problems such as global warming. Furthermore, fossil resources are not infinite, and will ultimately be depleted. To cope with these problems, the sustainable production of aromatic chemicals from renewable non-food biomass is urgent. With this in mind, the search for alternative methodologies to produce aromatic compounds using low-cost and environmentally friendly processes is becoming more and more important. Microorganisms are able to produce aromatic and aromatic-derivative compounds from sugar-based carbon sources. Metabolic engineering strategies as well as bioprocess optimization enable the development of microbial cell factories capable of efficiently producing aromatic compounds. This review presents current breakthroughs in microbial production of specialty aromatic and aromatic-derivative products, providing an overview on the general strategies and methodologies applied to build microbial cell factories for the production of these compounds. We present and describe some of the current challenges and gaps that must be overcome in order to render the biotechnological production of specialty aromatic and aromatic-derivative attractive and economically feasible at industrial scale.

Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.

The Biosynthesis of D-1,2,4-Butanetriol From d-Arabinose With an Engineered Escherichia coli.

D-1,2,4-Butanetriol (BT) has attracted much attention for its various applications in energetic materials and the pharmaceutical industry. Here, a synthetic pathway for the biosynthesis of BT from d-arabinose was constructed and optimized in Escherichia coli. First, E. coli Trans1-T1 was selected for the synthesis of BT. Considering the different performance of the enzymes from different organisms when expressed in E. coli, the synthetic pathway was optimized. After screening two d-arabinose dehydrogenases (ARAs), two d-arabinonate dehydratases (ADs), four 2-keto acid decarboxylases (ADXs), and three aldehyde reductases (ALRs), ADG from Burkholderia sp., AraD from Sulfolobus solfataricus, KivD from Lactococcus lactis IFPL730, and AdhP from E. coli were selected for the bio-production of BT. After 48 h of catalysis, 0.88 g/L BT was produced by the recombinant strain BT5. Once the enzymes were selected for the pathway, metabolic engineering strategy was conducted for further improvement. The final strain BT5ΔyiaEΔycdWΔyagE produced 1.13 g/L BT after catalyzing for 48 h. Finally, the fermentation conditions and characteristics of BT5ΔyiaEΔycdWΔyagE were also evaluated, and then 2.24 g/L BT was obtained after 48 h of catalysis under the optimized conditions. Our work was the first report on the biosynthesis of BT from d-arabinose which provided a potential for the large-scale production of d-glucose-based BT.

A paradigm shift towards production of sustainable bioenergy and advanced products from Cannabis/hemp biomass in Canada.

The global cannabis (Cannabis sativa) market was 17.7 billion in 2019 and is expected to reach up to 40.6 billion by 2024. Canada is the 2nd nation to legalize cannabis with a massive sale of $246.9 million in the year 2021. Waste cannabis biomass is managed using disposal strategies (i.e., incineration, aerobic/anaerobic digestion, composting, and shredding) that are not good enough for long-term environmental sustainability. On the other hand, greenhouse gas emissions and the rising demand for petroleum-based fuels pose a severe threat to the environment and the circular economy. Cannabis biomass can be used as a feedstock to produce various biofuels and biochemicals. Various research groups have reported production of ethanol 9.2–20.2 g/L, hydrogen 13.5 mmol/L, lipids 53.3%, biogas 12%, and biochar 34.6% from cannabis biomass. This review summarizes its legal and market status (production and consumption), the recent advancements in the lignocellulosic biomass (LCB) pre-treatment (deep eutectic solvents (DES), and ionic liquids (ILs) known as “green solvents”) followed by enzymatic hydrolysis using glycosyl hydrolases (GHs) for the efficient conversion efficiency of pre-treated biomass. Recent advances in the bioconversion of hemp into oleochemicals, their challenges, and future perspectives are outlined. A comprehensive insight is provided on the trends and developments of metabolic engineering strategies to improve product yield. The thermochemical processing of disposed-off hemp lignin into bio-oil, bio-char, synthesis gas, and phenol is also discussed. Despite some progress, barricades still need to be met to commercialize advanced biofuels and compete with traditional fuels.

Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketol-acid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe–S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol.

Living Bacteria-Based Immuno-Photodynamic Therapy: Metabolic Labeling of Clostridium butyricum for Eradicating Malignant Melanoma.

Due to the complexity, aggressiveness, and heterogeneity of malignant melanoma, it is difficult to eradicate the whole tumor through conventional treatment. Herein, a strategy of metabolic engineering labeled anaerobic oncolytic bacteria (Clostridium butyricum) is demonstrated to achieve the ablation of melanoma. In this system, the metabolic substrate of C. butyricum d‐alanine (d‐Ala) is first conjugated with a photosensitizer (TPApy) showing aggregation‐induced emission (AIE). The yielded metabolic substrate of d‐Ala‐TPAPy can be metabolically incorporated into bacterial peptidoglycan to form engineered C. Butyricum. Once the engineered C. butyricum is injected into melanoma, the bacteria can only proliferate in an anaerobic zone, stimulate the tumor immune microenvironment, and ablate the tumor hypoxia region. Following that, the relatively rich oxygen content in the peripheral area can induce the death of C. butyricum. The photosensitizer (PS) on the bacteria can subsequently exert a photodynamic effect in the oxygen‐rich region and further remove the melanoma residue under light irradiation. Prominent in vivo melanoma ablation results revealed that the engineering oncolytic bacteria can provide a promising regime for solid tumor eradication.

Construction and optimization of Saccharomyces cerevisiae for synthesizing forskolin.

Forskolin, one of the primary active metabolites of labdane-type diterpenoids, exhibits significant medicinal value, such as anticancer, antiasthmatic, and antihypertensive activities. In this study, we constructed a Saccharomyces cerevisiae cell factory that efficiently produced forskolin. First, a chassis strain that can accumulate 145.8 mg/L 13R-manoyl oxide (13R-MO), the critical precursor of forskolin, was constructed. Then, forskolin was produced by integrating CfCYP76AH15, CfCYP76AH11, CfCYP76AH16, ATR1, and CfACT1-8 into the 13R-MO chassis with a titer of 76.25 μg/L. We confirmed that cytochrome P450 enzymes (P450s) are the rate-limiting step by detecting intermediate metabolite accumulation. Forskolin production reached 759.42 μg/L by optimizing the adaptations between CfCYP76AHs, t66CfCPR, and t30AaCYB5. Moreover, multiple metabolic engineering strategies, including regulation of the target genes' copy numbers, amplification of the endoplasmic reticulum (ER) area, and cofactor metabolism enhancement, were implemented to enhance the metabolic flow to forskolin from 13R-MO, resulting in a final forskolin yield of 21.47 mg/L in shake flasks and 79.33 mg/L in a 5 L bioreactor. These promising results provide guidance for the synthesis of other natural terpenoids in S. cerevisiae, especially for those containing multiple P450s in their synthetic pathways. KEY POINTS: • The forskolin biosynthesis pathway was optimized from the perspective of system metabolism for the first time in S. cerevisiae. • The adaptation and optimization of CYP76AHs, t66CfCPR, and t30AaCYB5 promote forskolin accumulation, which can provide a reference for diterpenoids containing complex pathways, especially multiple P450s pathways. • The forskolin titer of 79.33 mg/L is the highest production currently reported and was achieved by fed-batch fermentation in a 5 L bioreactor.

Inhibiting tricin biosynthesis improves maize lignocellulose saccharification

Lignin is a technological bottleneck to convert polysaccharides into fermentable sugars, and different strategies of genetic-based metabolic engineering have been applied to improve biomass saccharification. Using maize seedlings grown hydroponically for 24 h, we conducted a quick non-transgenic approach with five enzyme inhibitors of the lignin and tricin pathways. Two compounds [3,4-(methylenedioxy)cinnamic acid: MDCA and 2,4-pyridinedicarboxylic acid: PDCA] revealed interesting findings on root growth, lignin composition, and saccharification. By inhibiting hydroxycinnamoyl-CoA ligase, a key enzyme of phenylpropanoid pathway, MDCA decreased the lignin content and improved saccharification, but it decreased root growth. By inhibiting flavone synthase, a key enzyme of tricin biosynthesis, PDCA decreased total lignin content and improved saccharification without affecting root growth. PDCA was three-fold more effective than MDCA, suggesting that controlling lignin biosynthesis with enzymatic inhibitors may be an attractive strategy to improve biomass saccharification.

Engineering Saccharomyces cerevisiae for efficient production of glucaric acid

As an important dicarboxylic acids existing in nature, glucaric acid has been widely used in medical, health, and polymer materials industry, therefore it is considered as one of the "top value-added chemicals from biomass". In this study, using Saccharomyces cerevisiae as a chassis microorganism, the effects of overexpression of myo-inositol transporter Itr1, fusional expression of inositol oxygenase MIOX4 and uronate dehydrogenase Udh, and down-expression of glucose-6-phosphate dehydrogenase gene ZWF1 on the glucaric acid production were investigated. The results showed that the yield of glucaric acid was increased by 26% compared with the original strain Bga-3 under shake flask fermentation after overexpressing myo-inositol transporter Itr1. The yield of glucaric acid was increased by 40% compared with Bga-3 strain by expressing the MIOX4-Udh fusion protein. On these basis, the production of glucaric acid reached 5.5 g/L, which was 60% higher than that of Bga-3 strain. In a 5 L fermenter, the highest yield of glucaric acid was 10.85 g/L, which was increased 80% compared with that of Bga-3 strain. The application of the above metabolic engineering strategy improved the pathway efficiency and the yield of glucaric acid, which may serve as a reference for engineering S. cerevisiae to produce other chemicals.

Multilevel interactions between native and ectopic isoprenoid pathways affect global metabolism in rice

Isoprenoids are natural products derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, these precursors are synthesized via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. The regulation of these pathways must therefore be understood in detail to develop effective strategies for isoprenoid metabolic engineering. We hypothesized that the strict regulation of the native MVA pathway could be circumvented by expressing an ectopic plastidial MVA pathway that increases the accumulation of IPP and DMAPP in plastids. We therefore introduced genes encoding the plastid-targeted enzymes HMGS, tHMGR, MK, PMK and MVD and the nuclear-targeted transcription factor WR1 into rice and evaluated the impact of their endosperm-specific expression on (1) endogenous metabolism at the transcriptomic and metabolomic levels, (2) the synthesis of phytohormones, carbohydrates and fatty acids, and (3) the macroscopic phenotype including seed morphology. We found that the ectopic plastidial MVA pathway enhanced the expression of endogenous cytosolic MVA pathway genes while suppressing the native plastidial MEP pathway, increasing the production of certain sterols and tocopherols. Plants carrying the ectopic MVA pathway only survived if WR1 was also expressed to replenish the plastid acetyl-CoA pool. The transgenic plants produced higher levels of fatty acids, abscisic acid, gibberellins and lutein, reflecting crosstalk between phytohormones and secondary metabolism.

Elucidation of Triacylglycerol Overproduction in the C4 Bioenergy Crop Sorghum bicolor by Constraint-Based Analysis.

Upregulation of triacylglycerols (TAGs) in vegetative plant tissues such as leaves has the potential to drastically increase the energy density and biomass yield of bioenergy crops. In this context, constraint-based analysis has the promise to improve metabolic engineering strategies. Here we present a core metabolism model for the C4 biomass crop Sorghum bicolor (iTJC1414) along with a minimal model for photosynthetic CO2 assimilation, sucrose and TAG biosynthesis in C3 plants. Extending iTJC1414 to a four-cell diel model we simulate C4 photosynthesis in mature leaves with the principal photo-assimilatory product being replaced by TAG produced at different levels. Independent of specific pathways and per unit carbon assimilated, energy content and biosynthetic demands in reducing equivalents are about 1.3 to 1.4 times higher for TAG than for sucrose. For plant generic pathways, ATP- and NADPH-demands per CO2 assimilated are higher by 1.3- and 1.5-fold, respectively. If the photosynthetic supply in ATP and NADPH in iTJC1414 is adjusted to be balanced for sucrose as the sole photo-assimilatory product, overproduction of TAG is predicted to cause a substantial surplus in photosynthetic ATP. This means that if TAG synthesis was the sole photo-assimilatory process, there could be an energy imbalance that might impede the process. Adjusting iTJC1414 to a photo-assimilatory rate that approximates field conditions, we predict possible daily rates of TAG accumulation, dependent on varying ratios of carbon partitioning between exported assimilates and accumulated oil droplets (TAG, oleosin) and in dependence of activation of futile cycles of TAG synthesis and degradation. We find that, based on the capacity of leaves for photosynthetic synthesis of exported assimilates, mature leaves should be able to reach a 20% level of TAG per dry weight within one month if only 5% of the photosynthetic net assimilation can be allocated into oil droplets. From this we conclude that high TAG levels should be achievable if TAG synthesis is induced only during a final phase of the plant life cycle.

SlWRKY35 positively regulates carotenoid biosynthesis by activating the MEP pathway in tomato fruit

Carotenoids are vital phytonutrients widely recognised for their health benefits. Therefore, it is vital to thoroughly investigate the metabolic regulatory network underlying carotenoid biosynthesis and accumulation to open new leads towards improving their contents in vegetables and crops. The outcome of our study defines SlWRKY35 as a positive regulator of carotenoid biosynthesis in tomato. SlWRKY35 can directly activate the expression of the 1-deoxy-d-xylulose 5-phosphate synthase (SlDXS1) gene to reprogramme metabolism towards the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, leading to enhanced carotenoid accumulation. We also show that the master regulator SlRIN directly regulates the expression of SlWRKY35 during tomato fruit ripening. Compared with the SlLCYE overexpression lines, coexpression of SlWRKY35 and SlLCYE can further enhance lutein production in transgenic tomato fruit, indicating that SlWRKY35 represents a potential target towards designing innovative metabolic engineering strategies for carotenoid derivatives. In addition to providing new insights into the metabolic regulatory network associated with tomato fruit ripening, our data define a new tool for improving fruit content in specific carotenoid compounds.

Generation and Characterization of Stable Redox-Reporter Mammalian Cell Lines of Biotechnological Relevance.

Cellular functions such as DNA replication and protein translation are influenced by changes in the intracellular redox milieu. Exogenous (i.e., nutrients, deterioration of media components, xenobiotics) and endogenous factors (i.e., metabolism, growth) may alter the redox homeostasis of cells. Thus, monitoring redox changes in real time and in situ is deemed essential for optimizing the production of recombinant proteins. Recently, different redox-sensitive variants of green fluorescent proteins (e.g., rxYFP, roGFP2, and rxmRuby2) have been engineered and proved suitable to detect, in a non-invasive manner, perturbations in the pool of reduced and oxidized glutathione, the major low molecular mass thiol in mammals. In this study, we validate the use of cytosolic rxYFP on two cell lines widely used in biomanufacturing processes, namely, CHO-K1 cells expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF) and HEK-293. Flow cytometry was selected as the read-out technique for rxYFP signal given its high-throughput and statistical robustness. Growth kinetics and cellular metabolism (glucose consumption, lactate and ammonia production) of the redox reporter cells were comparable to those of the parental cell lines. The hGM-CSF production was not affected by the expression of the biosensor. The redox reporter cell lines showed a sensitive and reversible response to different redox stimuli (reducing and oxidant reagents). Under batch culture conditions, a significant and progressive oxidation of the biosensor occurred when CHO-K1-hGM-CSF cells entered the late-log phase. Medium replenishment restored, albeit partially, the intracellular redox homeostasis. Our study highlights the utility of genetically encoded redox biosensors to guide metabolic engineering or intervention strategies aimed at optimizing cell viability, growth, and productivity.

Production of Caffeic Acid with Co-fermentation of Xylose and Glucose by Multi-modular Engineering in Candida glycerinogenes.

Caffeic acid (CA), a natural phenolic compound, has important medicinal value and market potential. In this study, we report a metabolic engineering strategy for the biosynthesis of CA in Candida glycerinogenes using xylose and glucose. The availability of precursors was increased by optimization of the shikimate (SA) pathway and the aromatic amino acid pathway. Subsequently, the carbon flux into the SA pathway was maximized by introducing a xylose metabolic pathway and optimizing the xylose assimilation pathway. Eventually, a high yielding strain CG19 was obtained, which reached a yield of 4.61 mg/g CA from mixed sugar, which was 1.2-fold higher than that of glucose. The CA titer in the 5 L bioreactor reached 431.45 mg/L with a yield of 8.63 mg/g of mixed sugar. These promising results demonstrate the great advantages of mixed sugar over glucose for high-yield production of CA. This is the first report to produce CA in C. glycerinogenes with xylose and glucose as carbon sources, which developed a promising strategy for the efficient production of high-value aromatic compounds.