Systems Metabolic Engineering Research Articles

Production of L-lactic acid from methanol by engineered yeast Pichia pastoris

Lactic acid (LA) serves as a widely used platform compound and has received significant attention as a raw material for synthesis of biodegradable polylactic acid. Currently, LA is mainly produced through microbial fermentation, but its high costs undermine its competitive advantage against other materials, necessitating the development of novel production routes. Methanol bioconversion represents an emerging low-carbon circular economy, where LA could become an outstanding representative product. This study successfully established an efficient methanol-based LA synthesis route in Pichia pastoris. Through systematic metabolic engineering strategies, including screening lactate dehydrogenase, modification of cofactor preference, blocking LA consumption pathway, and mitochondrial LA synthesis compartmentalization, 4.2 g/L L-LA was produced in fed-batch fermentation by using methanol as the sole carbon source. Through multi-dimensional and spatial engineering of enzyme, a cell factory was developed for efficient synthesis of L-LA, highlights the significant potential of the low-carbon synthesis route for L-LA via methanol bioconversion.

Metabolic Engineering of Corynebacterium glutamicum for the Production of the Four-Carbon Platform Chemicals γ-Hydroxybutyrate and γ-Butyrolactone.

γ-Hydroxybutyrate (GHB) is an important C4 platform chemical, serving as a crucial precursor for the synthesis of various bulk chemicals, including γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BDO). In this study, we report the systematic metabolic engineering of Corynebacterium glutamicum for the biological production of GHB from glucose via the introduction of a glutamate-derived pathway. We showed that C. glutamicum is a promising host for producing GHB due to its higher tolerance to GHB as compared to other chassis. By screening key enzymes capable of converting glutamate into GHB and blocking byproduct synthesis pathways, an engineered C. glutamicum strain was developed that achieved a GHB production titer of 30.6 g/L. Comparative transcriptome analysis was subsequently employed to identify previously uncharacterized aldehyde dehydrogenases responsible for succinate accumulation, and knockout of the corresponding genes led to an increased GHB titer of 33.7 g/L. Ultimately, the integration of a phosphoketolase-mediated nonoxidative glycolysis (NOG) pathway further enhanced GHB production, resulting in an accumulation of 38.3 g/L of GHB with a yield of 0.615 mol/mol glucose during batch fermentation. The GHB in the fermentation broth can be efficiently converted into GBL by acid treatment with a yield of 0.970 mol/mol.

Systematic metabolic engineering of Yarrowia lipolytica for efficient production of phytohormone abscisic acid

Abscisic acid (ABA) is an important phytohormone with diverse applications. It currently relies on the fermentation of Botrytis cinerea, which suffers from limited availability of genetic engineering tools. Here, Yarrowia lipolytica was engineered to enable de novo biosynthesis of ABA. To overcome the rate-limiting P450 enzymes, systematic engineering strategies were implemented. Firstly, the dissolved oxygen was increased to boost the activity of P450 enzymes. Secondly, the expansion of endoplasmic reticulum was implemented to improve the functional expression of P450 enzymes. Lastly, rate-limiting enzymes were assembled to facilitate substrate trafficking. Moreover, ABA production was further improved by strengthening the mevalonate pathway. Finally, the engineered strain produced 1221.45 mg/L of ABA in a 5-L bioreactor. The study provides effective approaches for alleviating rate-limiting P450 enzymes to enhance ABA production and achieve competitive industrial-level ABA production in Y. lipolytica.

De Novo Biosynthesis of Dihydroquercetin in Saccharomyces cerevisiae.

Dihydroquercetin is a vital flavonoid compound with a wide range of physiological activities. However, factors, such as metabolic regulation, limit the heterologous synthesis of dihydroquercetin in microorganisms. In this study, flavanone 3-hydroxylase (F3H) and flavanone 3'-hydroxylase (F3'H) were screened from different plants, and their co-expression in Saccharomyces cerevisiae was optimized. Promoter engineering and redox partner engineering were used to optimize the corresponding expression of genes involved in the dihydroquercetin synthesis pathway. Dihydroquercetin production was further improved through multicopy integration pathway genes and systems metabolic engineering. By increasing NADPH and α-ketoglutarate supply, the catalytic efficiency of F3'H and F3H was improved, thereby effectively increasing dihydroquercetin production (235.1 mg/L). Finally, 873.1 mg/L dihydroquercetin titer was obtained by fed-batch fermentation in a 5-L bioreactor, which is the highest dihydroquercetin production achieved through de novo microbial synthesis. These results established a pivotal groundwork for flavonoids synthesis.

Geranylgeraniol: Bio-based platform for teprenone, menaquinone-4, and α-tocotrienol synthesis

By utilizing the conformational selectivity of biosynthesis and the flexibility of chemical synthesis, researchers have formulated metabolic engineering-based semi-synthetic approaches that initiate with the final product’s structure and identify key biosynthesis intermediates. Nonetheless, these tailored semi-synthetic routes focused on end-products, neglecting the possibility of biobased intermediates as a platform for derivatization. To address this challenge, this studyproposed a novel strategy resembling chemosynthesis-style divergent exploration to amplify the significance of biobased intermediates, in the case of geranylgeraniol (GGOH). Using the novel bifunctional terpene synthase PTTC066 and systematic metabolic engineering modifications, the engineered yeast straindemonstrated high GGOH production levels (3.32 g/L, 0.039 g/L/h). This platformenabled the semi-synthesis of various pharmaceuticals, including the anti-ulcer drug teprenone, the osteoporosis treatment drug menaquinone-4, and introduced a novel route for synthesizingα-tocotrienol. This study offers a fresh outlook on semi-synthetic approaches, opening avenues for improvements, substitutions, and innovations in industrial production processes.

Systematic metabolic engineering for improved synthesis of perillic acid in Candida tropicalis

Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid’s precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis.Key points• Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis.• Perillic acid was first synthesized de novo in engineered microorganisms.• The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.

Metabolic engineering of Escherichia coli for thymidine production

Thymidine, as a crucial precursor of anti-AIDS drugs (e.g., zidovudine and stavudine), has wide application potential in the pharmaceutical industry. In this study, we introduced the thymidine biosynthesis pathway into the wild-type Escherichia coli MG1655 by systems metabolic engineering to improve the thymidine production in E. coli. Firstly, deoA, tdk, udp, rihA, rihB, and rihC were successively deleted to block the thymidine degradation pathway and salvage pathway in the wild-type E. coli MG1655. Then, the pyrimidine nucleoside operons from Bacillus subtilis F126 were introduced to enlarge the metabolic flux of the uridylic acid synthesis pathway. Finally, the expression of uridylate kinase, ribonucleoside diphosphate reductase, thymidine synthase, and 5'-nucleotidase in the thymidine biosynthesis pathway was optimized to enhance the metabolic flux from uridylic acid to thymidine. The engineered THY6-2 strain produced 11.10 g/L thymidine in a 5 L bioreactor with a yield of 0.04 g/g glucose and productivity of 0.23 g/(L·h). In this study, we constructed a strain that used glucose as the only carbon source for efficient production of thymidine and did not harbor plasmids, which provided a reference for the research on other pyrimidine nucleosides.

Advances in Flavonoid and Derivative Biosynthesis: Systematic Strategies for the Construction of Yeast Cell Factories.

Flavonoids, a significant group of natural polyphenolic compounds, possess a broad spectrum of pharmacological effects. Recent advances in the systematic metabolic engineering of yeast cell factories (YCFs) provide new opportunities for enhanced flavonoid production. Herein, we outline the latest research progress on typical flavonoid products in YCFs. Advanced engineering strategies involved in flavonoid biosynthesis are discussed in detail, including enhancing precursor supply, cofactor engineering, optimizing core pathways, eliminating competitive pathways, relieving transport limitations, and dynamic regulation. Additionally, we highlight the existing problems in the biosynthesis of flavonoid glucosides in yeast, such as endogenous degradation of flavonoid glycosides, substrate promiscuity of UDP-glycosyltransferases, and an insufficient supply of UDP-sugars, with summaries on the corresponding solutions. Discussions also cover other typical postmodifications like prenylation and methylation, and the recent biosynthesis of complex flavonoid compounds in yeast. Finally, a series of advanced technologies are envisioned, i.e., semirational enzyme engineering, ML/DL algorithn, and systems biology, with the aspiration of achieving large-scale industrial production of flavonoid compounds in the future.

Systems Metabolic Engineering to Elucidate and Enhance Intestinal Metabolic Activities of Escherichia coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is one of the most widely used probiotics to treat gastrointestinal diseases. Recently, many studies have engineered EcN to release therapeutic proteins to treat specific diseases. However, because EcN exhibits intestinal metabolic activities, it is difficult to predict outcomes after administration. In silico and fermentation profiles revealed mucin metabolism of EcN. Multiomics revealed that fucose metabolism contributes to the intestinal colonization of EcN by enhancing the synthesis of flagella and nutrient uptake. The multiomics results also revealed that excessive intracellular trehalose synthesis in EcN, which is responsible for galactose metabolism, acts as a metabolic bottleneck, adversely affecting growth. To improve the ability of EcN to metabolize galactose, otsAB genes for trehalose synthesis were deleted, resulting in the ΔotsAB strain; the ΔotsAB strain exhibited a 1.47-fold increase in the growth rate and a 1.37-fold increase in the substrate consumption rate relative to wild-type EcN.

Metabolic Engineering of Escherichia coli for Bioproduction of (R)-3-Hydroxybutyric Acid through a Three-Pronged Approach.

(R)-3-Hydroxybutyric acid (R-3HB) is an important chiral chemical with extensive applications in the agricultural, food, and chemical industries. The synthesis of R-3HB by microbial fermentation is of interest due to its remarkable stereoselectivity and economy. However, the low production of R-3HB failed to meet the needs of large-scale industrial production. In this study, an engineered strain for the efficient biosynthesis of R-3HB was constructed through a three-pronged approach encompassing biosynthetic pathway optimization, engineering of NADPH regenerators, and central metabolism regulation. The engineered strain Q5081 produced 75.7 g/L R-3HB, with a productivity of 1.26 g/L/h and a yield of 0.34 g/g glucose in fed-batch fermentation, showing the highest reported titer and productivity of R-3HB to date. We also performed transcriptome sequencing and annotation to illustrate the mechanism underlying the enhanced R-3HB production. The systematic metabolic engineering by a three-pronged approach demonstrated the feasibility of improving the biosynthesis, and the engineered strain Q5081 has the potential for widespread applications in the industrial production of R-3HB.

Efficient production of 22(R)-hydroxycholesterol via combination optimization of Saccharomyces cerevisiae.

22(R)-hydroxycholesterol (22(R)-HCHO) is a crucial precursor of steroids biosynthesis with various biological functions. However, the production of 22(R)-HCHO is expensive and unsustainable due to chemical synthesis and extraction from plants or animals. This study aimed to construct a microbial cell factory to efficiently produce 22(R)-HCHO through systems metabolic engineering. First, we tested 7-dehydrocholesterol reductase (Dhcr7s) and cholesterol C22-hydroxylases from different sources in Saccharomyces cerevisiae, and the titer of 22(R)-HCHO reached 128.30mg L-1 in the engineered strain expressing Dhcr7 from Columba livia (ClDhcr7) and cholesterol 22-hydroxylase from Veratrum californicum (VcCyp90b27). Subsequently, the 22(R)-HCHO titer was significantly increased to 427.78mg L-1 by optimizing the critical genes involved in 22(R)-HCHO biosynthesis. Furthermore, hybrid diploids were constructed to balance cell growth and 22(R)-HCHO production and to improve stress tolerance. Finally, the engineered strain produced 2.03g L-1 of 22(R)-HCHO in a 5-L fermenter, representing the highest 22(R)-HCHO titer reported to date in engineered microbial cell factories. The results of this study provide a foundation for further applications of 22(R)-HCHO in various industrially valuable steroids.

Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering

Vitamin B5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel “PEACES” system by (1) expressing the NAD + kinase gene ppnk from Clostridium glutamicum and the NADP + -dependent gapCcae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.

Systems metabolic engineering of Escherichia coli for high-yield production of Para-hydroxybenzoic acid

Para-hydroxybenzoic acid (PHBA) is extensively used as an additive in the food and cosmetics industries, significantly enhancing product shelf life and stability. While microbial fermentation offers an environment-friendly and sustainable method for producing PHBA, the titer and productivity are limited due to product toxicity and complex metabolic flux distributions. Here, we initially redesigned a L-phenylalanine-producing Escherichia coli by employing rational metabolic engineering strategies, resulting in the production of PHBA reached the highest reported level of 14.17 g/L. Subsequently, a novel accelerated evolution system was devised comprising deaminase, the alpha subunit of RNA polymerase, an uracil-DNA glycosylase inhibitor, and the PHBA-responsive promoter PyhcN. This system enabled us to obtain a mutant strain exhibiting a 47% increase in the half-inhibitory concentration (IC50) for PHBA within 15 days. Finally, the evolved strain achieved a production of 21.35 g/L PHBA in a 5-L fermenter, with a yield of 0.19 g/g glucose and a productivity rate of 0.44 g/L/h. This engineered strain emerges as a promising candidate for industrial production of PHBA through an eco-friendly approach.

Metabolic engineering of Pichia pastoris for overproduction of cis-trans nepetalactol

Monoterpene indole alkaloids (MIAs) are a group of plant-derived natural products with high-value medicinal properties. However, their availability for clinical application is limited due to challenges in plant extraction. Microbial production has emerged as a promising strategy to meet the clinical demands for MIAs. The biosynthetic pathway of cis-trans nepetalactol, which serves as the universal iridoid scaffold for all MIAs, has been successfully identified and reconstituted. However, bottlenecks and challenges remain to construct a high-yielding platform strain for cis-trans nepetalactol production, which is vital for subsequent MIAs biosynthesis. In the present study, we focused on engineering of Pichia pastoris cell factories to enhance the production of geraniol, 8-hydroxygeraniol, and cis-trans nepetalactol. By targeting the biosynthetic pathway from acetyl-CoA to geraniol in both peroxisomes and cytoplasm, we achieved comparable geraniol titers in both compartments. Through protein engineering, we found that either G8H or CPR truncation increased the production of 8-hydroxygeraniol, with a 47.8-fold and 14.0-fold increase in the peroxisomal and cytosolic pathway strain, respectively. Furthermore, through a combination of dynamical control of ERG20, precursor and cofactor supply engineering, diploid engineering, and dual subcellular compartmentalization engineering, we achieved the highest ever reported production of cis-trans nepetalactol, with a titer of 4429.4 mg/L using fed-batch fermentation in a 5-L bioreactor. We anticipate our systematic metabolic engineering strategies to facilitate the development of P. pastoris cell factories for sustainable production of MIAs and other plant natural products.

Efficient Fermentative Production of β-Alanine from Glucose through Multidimensional Engineering of Escherichia coli.

β-Alanine, a valuable β-type amino acid, is experiencing increased demand due to its multifaceted applications in food flavoring, nutritional supplements, pharmaceuticals, and the chemical industry. Nevertheless, the sustainable biosynthesis of β-alanine currently faces challenges due to the scarcity of robust strains, attributed to the complexities of modulating multiple genes and the inherent physiological constraints. Here, systems metabolic engineering was implemented in Escherichia coli to overcome these limitations. First, an efficient l-aspartate-α-decarboxylase (ADC) was recruited for β-alanine biosynthesis. To conserve phosphoenolpyruvate flux, we subsequently modified the endogenous glucose assimilation system by inactivating the phosphotransferase system (PTS) and introducing an alternative non-PTS system, which increased β-alanine production to 1.70 g/L. The supply of key precursors, oxaloacetate and l-aspartate, was synergistically improved through comprehensive modulation, including strengthening main flux and blocking bypass metabolism, which significantly increased the β-alanine titer to 3.43 g/L. Next, the expression of ADC was optimized by promoter and untranslated region (UTR) engineering. Further transport engineering, which involved disrupting β-alanine importer CycA and heterologously expressing β-alanine exporter NCgI0580, improved β-alanine production to 8.48 g/L. Additionally, corn steep liquor was used to develop a cost-effective medium. The final strain produced 74.03 g/L β-alanine with a yield of 0.57 mol/mol glucose during fed-batch fermentation.

Systematic metabolic engineering of Zymomonas mobilis for β-farnesene production.

Zymomonas mobilis is an ethanologenic bacterium that can produce hopanoids using farnesyl pyrophosphate (FPP), which can be used as the precursor by β-farnesene synthase for β-farnesene production. To explore the possibility and bottlenecks of developing Z. mobilis for β-farnesene production, five heterologous β-farnesene synthases were selected and screened, and AaBFS from Artemisia annua had the highest β-farnesene titer. Recombinant strains with AaBFS driven by the strong constitutive promoter Pgap (Pgap-AaBFS) doubled its β-farnesene production to 25.73 ± 0.31mg/L compared to the recombinant strain with AaBFS driven by Ptet (Ptet-AaBFS), which can be further improved by overexpressing the Pgap-AaBFS construct using the strategies of multiple plasmids (41.00 ± 0.40mg/L) or genomic multi-locus integration (48.33 ± 3.40mg/L). The effect of cofactor NADPH balancing on β-farnesene production was also investigated, which can be improved only in zwf-overexpressing strains but not in ppnK-overexpressing strains, indicating that cofactor balancing is important and sophisticated. Furthermore, the β-farnesene titer was improved to 73.30 ± 0.71mg/L by overexpressing dxs, ispG, and ispH. Finally, the β-farnesene production was increased to 159.70 ± 7.21mg/L by fermentation optimization, including the C/N ratio, flask working volume, and medium/dodecane ratio, which was nearly 13-fold improved from the parental strain. This work thus not only generated a recombinant β-farnesene production Z. mobilis strain but also unraveled the bottlenecks to engineer Z. mobilis for farnesene production, which will help guide the future rational design and construction of cell factories for terpenoid production in non-model industrial microorganisms.

Combinatorial Metabolic Engineering for Improving Betulinic Acid Biosynthesis in Saccharomyces cerevisiae.

Betulinic acid (BA) is a lupane-type triterpenoid with potent anticancer and anti-HIV activities. Its great potential in clinical applications necessitates the development of an efficient strategy for BA synthesis. This study attempted to achieve efficient BA biosynthesis in Saccharomyces cerevisiae using systematic metabolic engineering strategies. First, a de novo BA biosynthesis pathway in S. cerevisiae was constructed, which yielded a titer of 14.01 ± 0.21 mg/L. Then, by enhancing the BA synthesis pathway and dynamic inhibition of the competitive pathway, a greater proportion of the metabolic flow was directed toward BA synthesis, achieving a titer of 88.07 ± 5.83 mg/L. Next, acetyl-CoA and NADPH supply was enhanced, which increased the BA titer to 166.43 ± 1.83 mg/L. Finally, another BA synthesis pathway in the peroxisome was constructed. Dual regulation of the peroxisome and cytoplasmic metabolism increased the BA titer to 210.88 ± 4.76 mg/L. Following fed-batch fermentation process modification, the BA titer reached 682.29 ± 8.16 mg/L. Overall, this work offers a guide for building microbial cell factories that are capable of producing terpenoids with efficiency.

De novo production of bioactive sesterterpenoid ophiobolins in Saccharomyces cerevisiae cell factories

BackgroundSesterterpenoids are rare species among the terpenoids family. Ophiobolins are sesterterpenes with a 5-8-5 tricyclic skeleton. The oxidized ophiobolins exhibit significant cytotoxic activity and potential medicinal value. There is an urgent need for large amounts of ophiobolins supplication for drug development. The synthetic biology approach has been successfully employed in lots of terpene compound production and inspired us to develop a cell factory for ophiobolin biosynthesis.ResultsWe developed a systematic metabolic engineering strategy to construct an ophiobolin biosynthesis chassis based on Saccharomyces cerevisiae. The whole-cell biotransformation methods were further combined with metabolic engineering to enhance the expression of key ophiobolin biosynthetic genes and improve the supply of precursors and cofactors. A high yield of 5.1 g/L of ophiobolin F was reached using ethanol and fatty acids as substrates. To accumulate oxidized ophiobolins, we optimized the sources and expression conditions for P450-CPR and alleviated the toxicity of bioactive compounds to cells through PDR engineering. We unexpectedly obtained a novel ophiobolin intermediate with potent cytotoxicity, 5-hydroxy-21-formyl-ophiobolin F, and the known bioactive compound ophiobolin U. Finally, we achieved the ophiobolin U titer of 128.9 mg/L.ConclusionsWe established efficient cell factories based on S. cerevisiae, enabling de novo biosynthesis of the ophiobolin skeleton ophiobolin F and oxidized ophiobolins derivatives. This work has filled the gap in the heterologous biosynthesis of sesterterpenoids in S. cerevisiae and provided valuable solutions for new drug development based on sesterterpenoids.

Enhancing erythromycin production in Saccharopolyspora erythraea through rational engineering and fermentation refinement: A Design-Build-Test-Learn approach.

Industrial production of bioactive compounds from actinobacteria, such as erythromycin and its derivatives, faces challenges in achieving optimal yields. To this end, the Design-Build-Test-Learn (DBTL) framework, a systematic metabolic engineering approach, was employed to enhance erythromycin production in Saccharopolyspora erythraea (S. erythraea) E3 strain. A genetically modified strain, S. erythraea E3-CymRP21-dcas9-sucC (S. erythraea CS), was developed by suppressing the sucC gene using an inducible promoter and dcas9 protein. The strain exhibited improved erythromycin synthesis, attributed to enhanced precursor synthesis and increased NADPH availability. Transcriptomic and metabolomic analyses revealed altered central carbon metabolism, amino acid metabolism, energy metabolism, and co-factor/vitamin metabolism in CS. Augmented amino acid metabolism led to nitrogen depletion, potentially causing cellular autolysis during later fermentation stages. By refining the fermentation process through ammonium sulfate supplementation, erythromycin yield reached 1125.66mg L-1, a 43.5% increase. The results demonstrate the power of the DBTL methodology in optimizing erythromycin production, shedding light on its potential for revolutionizing antibiotic manufacturing in response to the global challenge of antibiotic resistance.

Systems metabolic engineering of Corynebacterium glutamicum to assimilate formic acid for biomass accumulation and succinic acid production

Formate as an ideal mediator between the physicochemical and biological realms can be obtained from electrochemical reduction of CO2 and used to produce bio-chemicals. Yet, limitations arise when employing natural formate-utilizing microorganisms due to restricted product range and low biomass yield. This study presents a breakthrough: engineered Corynebacterium glutamicum strains (L2-L4) through modular engineering. L2 incorporates the formate-tetrahydrofolate cycle and reverse glycine cleavage pathway, L3 enhances NAD(P)H regeneration, and L4 reinforces metabolic flux. Metabolic modeling elucidates C1 assimilation, guiding strain optimization for co-fermentation of formate and glucose. Strain L4 achieves an OD600 of 0.5 and produces 0.6 g/L succinic acid. 13C-labeled formate confirms C1 assimilation, and further laboratory evolution yields 1.3 g/L succinic acid. This study showcases a successful model for biologically assimilating formate in C. glutamicum that could be applied in C1-based biotechnological production, ultimately forming a formate-based bioeconomy.