Establishment of the REMBAC-cassette, a rapid, efficient and manifold BacMam tool for recombinant protein expression
Efficient recombinant protein production requires mammalian stable cell lines or often relies on inefficient transfection processes. Baculoviral transduction of mammalian cells (BacMam) offers cost-effective and robust gene transfer and straightforward scalability. The advantages over conventional approaches are, no need of high biosafety level laboratories, efficient transduction of various cell types and transfer of large transgenes into host cells. In our study, we aim to develop a high expression cassette to increase yields of baculoviral transduction. The establishment follows a sequential approach by first identifying the strongest promoter, followed by intron and WPRE sequences as enhancer elements for transcription and translation. The resulting REMBAC-cassette was compared to conventional transfection in suspension and adherent cells. Irrespective of the cell line, transduction reached nearly 100 % efficiency and led to almost 10-fold increases of gene expression levels. We confirmed these results in larger scale with batch and fed-batch cultivations. Finally, expression of different soluble proteins with high degrees of complexity confirmed the versatility of our established cassette. Overall, the REMBAC-cassette incorporated into the BacMam platform is a manifold tool offering advantages over standard transfection, in the scalability, efficiency and gene expression, which results in higher yields, shorter cultivation times and consequently cost-effective production processes.
Converting multiple hydrophobic aromatic plastic monomers into a single water-soluble substrate to increase bioavailability for the synthesis of polyhydroxyalkanoates by bacteria using batch, fed batch and continuous cultivation
We demonstrate the proof of concept of increasing the bioavailability of carbon substrates, derived from plastic waste, for their conversion to the biodegradable polymer polyhydroxyalkanoate [PHA] by bacteria and test various approaches to PHA accumulation through batch, fed batch and continuous culture. Styrene, ethylbenzene, and toluene are produced from the pyrolysis of mixed plastic waste (Kaminsky, 2021; Miandad et al., 2017), but they are volatile and poorly soluble in water making them difficult to work with in aqueous fermentation systems. By chemically converting these aromatic compounds to benzoic acid, and subsequently to its sodium salt, we increased the solubility and reduced the volatility of the substrate supplied to Pseudomonas putida CA-3 to accumulate polyhydroxyalkanoates. 1 L scale batch, fed batch, and continuous fermentations were carried out; the fed batch fermentation resulted in the maximum volumetric PHA productivity of 61.67 ± 7.34 mg L h; while batch and continuous, at a dilution rate, d = 0.2 h, fermentations resulted in 13.30 ± 0.01 and 4.06 ± 0.01 mg L h of PHA respectively.
High-yield soluble production of recombinant β-keratin from Gallus gallus feathers using an experimental design approach
The search for new non-animal textile materials has increased yearly as environmental awareness and veganism continue to spread, driving the development of greener fabrics. Concurrently, β-keratin, a fibrous, resistant, and insoluble protein shows great potential for producing innovative biomaterials. However, β-keratin is naturally abundant in animal feathers. Therefore, the recombinant production of β-keratin from Gallus gallus feathers was proposed using a strategy of parallel expression in different vectors. Statistical tools of experimental design were employed to improve the production of soluble biosynthetic keratin. It was shown that β-keratins fused to HisMBP had better performance regarding soluble expression. In addition, the optimized regions for the values of induction temperature, induction time, and induction absorbance were obtained. As a result, a yield of 185.3 ± 1.4 mg/L of soluble HisMBP-Chr2.FK4 was achieved, representing the highest yield reported to date.
Effects of ORF14 gene on melanin expression, fermentation conditions and properties of melanin production in modified strains
Melanin with antioxidant and antibacterial properties can be used in food, cosmetics, biotechnology, and other fields, but its insolubility become a main challenge hindering for its application. In this study, water-soluble melanin produced by the novel species Streptomyces vilmorinianum YP1 was characterized using scanning electron microscopy (SEM), UVvisible spectroscopy (with an absorption peak at 220 nm), and Fourier transform infrared (FTIR) spectroscopy. The glycosyltransferase gene ORF14 was knocked out, which improved the production of water-soluble melanin by inhibiting competitive pathway. In order to further enhance production of melanin, PlackettBurman and response surface methodology statistical design was employed to screen for key factors and determine the optimal combination. The maximum melanin production (4.00 g/L) was obtained under the conditions: amylodextrine concentration of 40 g/L, soya peptone concentration of 7 g/L, tryptone concentration of 5 g/L, NaCl concentration of 5.4 g/L, pH of 6.7 and temperature of 36 °C for 180 h. The physicochemical properties and bioactivity of melanin were further investigated, revealing that melanin had a good stability across a pH range of 4-12, antioxidant (with a survival rate of over 85 %), and resistance to reducing agents (with a survival rate of over 99 %). The results underscored that S. vilmorinianum YP1 is a promising candidate for water-soluble melanin production.
Improved biosynthesis of tyrosol by epigenetic modification-based regulation and metabolic engineering in Saccharomyces cerevisiae
Aromatic amino acids and their derivatives are high value chemicals widely used in food, pharmaceutical and feed industries. Current preparation methods for aromatic amino acid products are fraught with limitations. In this study, the efficient biosynthesis of aromatic amino acid compound tyrosol was investigated by epigenetic modification-based regulation and optimization of the biosynthetic pathway of aromatic amino acids. The production of tyrosol was significantly improved by the overexpression of mA modification writer Ime4 and reader Pho92, and the positive regulator Gcr2. Introduction of Bbxfpk and deletion of Gpp1 further improved tyrosol production. Then the feedback inhibition of the shikimate pathway was relieved by the mutants Aro4 and Aro7. The final tyrosol producing engineered strain was constructed by the deletion of PHA2, replacement of the native promoter of ARO10 with the strong promoter PGK1p, and introduction of tyrosine decarboxylase PcAAS. In the background of mA modification regulation, this strain ultimately produced 954.69 ± 43.72 mg/L of tyrosol, promoted by 61.7-fold in shake-flask fermentation.
Customized design of host-independent T7 expression system (HITES) for a broad host range
Efficient methods and universal DNA elements are eagerly required for the expression of proteins and the production of target chemicals in synthetic biology and metabolic engineering. This paper develops a customized-design approach by utilizing the host-independent T7 expression system (HITES), which facilitates the rational design and rapid construction of T7 expression systems. Firstly, the EL (Upper-limit value of initial enzyme activity) value is discovered to play a pivotal factor in the successful construction of the T7 expression system, different host strains exhibit varying EL values, and this study presents a method to measure the EL values. Secondly, E. coli DH5α is chosen as the host strain, and it demonstrates that various strategies to modulate T7 RNA polymerase activity can efficiently construct the HITES T7 expression system in E. coli DH5α under the guidance of EL. Lastly, the customized-design of HITES enables the efficient expression of sfGFP and D-MIase proteins across 13 host strains, guided by EL values. This customized-design method of HITES offers a streamlined pathway for T7 system construction across a broad range of hosts and serves as an enabling tool for synthetic biology, metabolic engineering, and enzyme engineering.
Immobilization of glycosyltransferase into a hydrophilic metal-organic framework for efficient biosynthesis of chondroitin sulfate
Chondroitin sulfate (CS) is a structurally complex anionic polysaccharide widely used in medical, cosmetic and food applications. Enzymatic catalysis is an important strategy for synthesizing CS with uniform chain lengths and well-defined structures. However, the industrial application of glycosyltransferases is hindered by limitations such as low expression yields, poor stability, and challenges in reuse. We developed a mild and rapid one-step synthetic method for the efficient immobilization of chondroitin synthase (KfoC). The resulting KfoC@ZIF-90 composite exhibits high catalytic activity, thermal stability, and pH adaptability. Notably, KfoC@ZIF-90 exhibited 5-fold enhanced thermal stability at 40°C and retained 86 % relative activity at pH 10, while also maintaining 90 % activity in organic solvents, surpassing the performance of free KfoC. Molecular docking analysis revealed that the binding capability of encapsulated KfoC with substrate was stronger than that of free KfoC, thereby improving catalytic performance. Furthermore, KfoC@ZIF-90 can be easily separated from the reaction solution by centrifugation, simplifying product isolation and purification while enabling enzyme reuse. These attributes significantly enhance operability and reduce processing costs, making enzymatic CS synthesis more feasible for industrial applications.
Ni-induced selective precipitation of His-tagged recombinant proteins shortens purification time while maintaining high yield
Nickel-NTA affinity chromatography is the current standard method for purifying His-tagged recombinant proteins. However, this process involves repetitive tasks, can be time-consuming, and reduces protein yield. Here, we present a simple, fast, and handy method for purifying His-tagged proteins using free Ni²⁺. This approach allows the fractional precipitation of His-tagged proteins directly from E. coli cell lysates. We successfully applied this Ni²⁺-based method to purify three His₆-tagged recombinant proteins overexpressed in E. coli. We found that Ni²⁺ at a final concentration of as low as 1 mM precipitates the His-tagged proteins with near-complete specificity as confirmed by SDS-PAGE analysis. The Ni²-precipitated proteins were dissolved by adding 10 % acetic acid and further purified by reverse-phase HPLC. The final yields were between 3.5 and 8.0 mg per 200 mL culture, similar to or even higher than purification using conventional Ni-NTA chromatography. The purified proteins exhibited natively folded characteristics, as assessed by CD, SLS, and DLS, and binding activity, as assessed by ELISA and BLI, demonstrating the method's potential in both small and large-scale settings.
Engineering silica nanocoated whole-cell asymmetric biocatalyst for efficient preparation of a key chiral intermediate of (S)-Rivastigmine
In our previous study, the whole cells containing an aldo-keto reductase (yhdN) and glucose dehydrogenase (GDH) were constructed and applied in a stereoselective carbonyl reduction reaction to prepare (S)-NEMCA-HEPE, being a key chiral intermediate of (S)-Rivastigmine which is widely prescribed for the treatment of Alzheimer's disease. Although the conversion and enantiomeric excess (e.e.) could reach to 78.2 % and 99 %, respectively, ionic liquid as an additive was required to improve the permeability of cell membrane. To further simplify the reaction, the molecular docking and saturation mutagenesis technology were used here to obtain an activity-improved yhdN variant such as G19A. And then, both excellent conversion and e.e. of 99 % for (S)-NEMCA-HEPE could be achieved within 40 min by using only G19A-GDH whole cell as a catalyst without any additive. However, the use of the whole cells still faces the issues of poor operation stability and adverse application prospect. Subsequently, a hydrophobic "cell-in-shell" complex of G19A-GDH@O-Silica was constructed by using a silica nanocoated technology. The obtained G19A-GDH@O-Silica exhibited an excellent conversion towards the asymmetric carbonyl reduction, and a good tolerance in changing thermal, pH, and storage environmental. Giving 76.3 % of reaction conversion even after the 11th cycle of reuse, indicated that G19A-GDH@O-Silica also possessed ideal recyclability. The aim of this study is to provide a rapid, and cost-effective nanocoated whole-cell biocatalyst for efficient preparation of (S)-NEMCA-HEPE. The simplicity and robustness of the immobilization approach may become a powerful tool to utilize whole-cell catalysts towards organic catalysis.
Structure-guided mining of stereoselective reductive aminases for biocatalytic stereodivergent synthesis of chiral piperidinamine and derivatives
Chiral azacyclic amine derivatives occupy a vital role of nitrogen-containing compounds, due to serve as foundational motifs in numerous pharmaceuticals and bioactive substances. Novel complementary enantioselective reductive aminases IRED9 and IRED11 were unveiled through comprehensive gene mining from Streptomyces viridochromogenes and Micromonospora echinaurantiaca, respectively, which both demonstrated enantiomeric excess (ee) values and conversion ratios of up to 99 % towards N-Boc-3-pyridinone (NBPO) and cyclopropylamine. IRED9 exhibited the highest activity at pH 8.0 and 45 °C,while IRED11 have optimal conditions at pH 8.0 and 50 °C. A variety of amine donors and ketones could be converted by IRED9 and IRED11 for asymmetric synthesis of piperidinamine and derivatives with complementary enantioselectivity. Through preparative-scale synthesis of (S)- and (R)-3-piperidinamine, IRED9 and IRED11 demonstrate substrate loadings of 120 g·L and 40 g·L with 98 % yield and 99 % ee, respectively. The space time yield (STY) reached 142.7 g·Ld and 47.1 g·Ld for the S enantiomer and R enantiomer, respectively. Interaction analysis indicated the substrate orientation and strong charge attraction interaction are vital factors for enantioselectivity of IREDs. This study unveils novel enantioselective reductive aminases for stereodivergent synthesis of piperidinamine and derivatives at high substrate loading.
Construction and characterization of a mutant library for the P constitutive promoter in lactic acid bacteria
Promoters are crucial elements for controlling gene expression in cells, yet lactic acid bacteria (LAB) often lack a diverse set of available constitutive promoters with quantitative characterization. To enrich the LAB promoter library, this study focused on the known strong constitutive promoter P in LAB. Through error-prone PCR and dNTP analog-induced random mutagenesis, a library of 247 mutants of P was generated by using the red fluorescent protein (RFP) fluorescence intensity as a high-throughput screening indicator in Streptococcus thermophilus. The activity of P mutants varied from 0.01 to 3.63 times that of P. Similar trends of promoter strength were observed in Lactobacillus plantarum and Lactococcus lactis, but significant differences in Escherichia coli, indicating the library's specificity to LAB. To assess the application potential of this P library, seven promoters with different strengths (0.28-2.58) were selected. The mutant promoters significantly enhanced the enzyme activities of superoxide dismutase (SOD), β-glucuronidase (GusA), and β-galactosidase (β-gal) in S. thermophilus. Notably, the mutant P expressing SOD exhibited an enzyme activity of 382.9 U/mg, which was 1.65 times higher than the control (P). Similarly, the expression of GusA and β-gal were 1.82 and 1.28 times higher than those of P, respectively. This study provided a set of significantly different P constitutive promoter mutant elements for the first time, laying the foundation for metabolic engineering and synthetic biology applications in LAB.
Investigating subpopulation dynamics in clonal CHO-K1 cells with single-cell RNA sequencing
Chinese Hamster Ovary (CHO) cells produce monoclonal antibodies and other biotherapeutics at industrial scale. Despite their ubiquitous nature in the biopharmaceutical industry, little is known about the behaviors of individual transfected clonal CHO cells. Most CHO cells are assessed on their stability, their ability to produce the protein of interest over time. But CHO cells have primarily been studied in bulk, instead assuming that these bulk samples are homogenous because of presumed genetic clonality across the sample. This does not address cellular heterogeneity in these ostensibly clonal cells. These variable stability phenotypes may reflect heterogeneity within the clonal samples. In this study, we performed single-cell RNA sequencing on two clonal CHO-K1 cell populations with different stability phenotypes over a 90 day culture period. Our data showed that the instability of one of the clone's gene expression was due in part to the emergence of a low-producing subpopulation in the aged samples. This low-producing subpopulation did not exhibit markers of cellular stress which were expressed in the higher-producing populations. Further multiomic investigation should be performed to better characterize this heterogeneity.
Fecal microbiota transplantation combined with inulin promotes the development and function of early immune organs in chicks
Modern management of chicks hinders the vertical transmission of intestinal microbiota, which is closely related to immunity. Inulin is a substrate that can be utilized by the microbiota. This study aimed to determine whether fecal microbiota transplantation (FMT) combined with inulin played a "1 + 1 > 2" role in enhancing the development and function of immune organs. Chicks were treated with 1 % inulin and/or fecal microbiota suspension on days 1-6. The growth performance, immune organ development, and immune indicators were evaluated on days 7, 14, and 21. Results showed that the combination of FMT and inulin significantly increased the immune organ index on day 7 and promoted the morphological structure and the expression of proliferating cell nuclear antigen (PCNA) in immune organs on days 7, 14, and 21. Each treatment increased the gene expression of interferon-gamma (IFN-γ), interleukin-4 (IL-4), interleukin-2 (IL-2), B cell-activating factor receptor (BAFFR), B cell linker (BLNK), C-X-C Motif Chemokine Ligand 12 (CXCL12), C-X-C Motif Chemokine Receptor 4 (CXCR4), and Biotin (Bu-1) to varying degrees. FMT combined with inulin significantly increased the expression of IgA-positive cells on days 7 and 14. In conclusion, the synergistic effect of FMT and inulin had beneficial impacts on the development and function of immune organs.
Investigating the metabolic load of monoclonal antibody production conveyed to an inducible CHO cell line using a transfer-rate online monitoring system
Shake flasks are a foundational tool in early process development by allowing high throughput exploration of the design space. However, lack of online data at this scale can hamper rapid decision making. Oxygen transfer rate (OTR) monitoring has been readily applied as an online process characterization tool at the benchtop bioreactor scale. Recent advances in modern sensing technology have allowed OTR monitoring to be available at the shake flask level. It is now possible to multiplex time-of-action (e.g., Induction, temperature shift, pH shift, feeding initiation, point of harvest) characterization studies by relying on careful analysis of OTR profile kinetics. As a result, there is potential to save time and capital expenditures while exploring process intensification studies though accurate and physiologically relevant online data. In this article, we detail the application of OTR monitoring to characterize the impact that recombinant protein production has on an inducible CHO cell line expressing Palivizumab. We then test out time-of-action studies to intensify protein production outcomes. We observe that recombinant protein expression causes a metabolic load that diminishes potential biomass growth. As a result, when compared to a control standard process, delaying induction and temperature shift has the potential to improve viable cell densities (VCD) by 2-fold thus increasing recombinant protein yield by over 30 %. The study also demonstrates that OTR can serve as a useful tool to detect cessation of exponential growth. Consequently, time-of-action points that are characteristic of inducible systems can be formulated accurately and reliably to maximize production performance.
Development of a highly efficient microbial fermentation process of recombinant Escherichia coli for GABA production from glucose
This study was aimed to develop a highly productive microbial fermentation process for gamma-aminobutyric acid (GABA) production from glucose. For this, an efficient GABA-producing E. coli strain was firstly developed through metabolic engineering with a strategy of increasing the flux of GABA biosynthetic pathway and deleting or repressing the GABA shunt pathways that compete with GABA biosynthesis. According to this strategy, three metabolically engineered E. coli strains of GTB, GTS, and A1S1 were constructed, and through batch cultivation of these strains, E. coli GTS was ultimately selected as the most efficient GABA-producing strain. From flask cultures, E. coli GTS was found to produce 3.96 g/L of GABA, a titer 2.1 times or 17 % higher than that produced by E. coli GTB or E. coli A1S1, respectively. To maximize GABA production from glucose, pH-stat fed-batch culture conditions of the E. coli GTS were optimized in a one-factor-at-a-time manner. Fed-batch cultivation of the E. coli GTS under optimal conditions resulted in the highest GABA production performance with a concentration of 85.9 g/L and a volumetric productivity of 2.37 g/L/h. This result shows that the microbial fermentation process developed in this study has outstanding potential for the mass production of GABA.
Hemp tea waste-immobilized lipase for the synthesis of alkyl oleates in solvent free systems
This study explores the immobilization of lipase from Candida rugosa (CRL) on hemp tea waste to catalyze the esterification of oleic acid with primary aliphatic C2-C12 alcohols in a solvent-free system. The immobilization method employed was adsorption, chosen for its simplicity, low cost, and ability to preserve enzyme activity. The esterification of undecanoic acid, lauric acid, and oleic acid with alcohols of varying chain lengths (ethanol, 1-propanol, 1-butanol, 1-octanol, 1-decanol, and 1-dodecanol) was studied. The esterification efficiency was found to be influenced by the type of alcohol, the molar ratio of oleic acid to alcohol, and the water content in the reaction medium. The highest conversions were achieved with ethanol (23 % conversion with 18.5 % water and a molar ratio of 1:5) and 1-decanol (30 % conversion with no added water and a molar ratio of 1:2). Comparatively, the soluble lipase achieved slightly higher conversion of oleic acid using decanol (38 %), indicating that the performance may be attributed to origin of lipase, rather than the immobilization procedure. The study demonstrated that the immobilization of lipase on hemp tea waste did not hinder enzyme activity. Additionally, the biocatalyst developed can function in a completely solvent-free system, offering a green solution by repurposing waste materials for industrial ester production.
Efficient Spermidine Production Using a Multi-Enzyme Cascade System Utilizing Methionine Adenosyltransferase from Lactobacillus fermentum with Reduced Product Inhibition and Acidic pH Preference
Methionine adenosyltransferases (MATs; EC 2.5.1.6) are key enzymes that catalyze a crucial step in the spermidine biosynthesis pathway. Due to MAT's significant product inhibition, S-adenosylmethionine (SAM) and spermidine production faces challenges. We evaluated MATs from 20 lactic acid bacteria (LAB) to identify enzymes with acidic preference and lower susceptibility to product inhibition. Lactobacillus fermentum's MAT (LfMAT) emerged as a candidate with desirable characteristics. LfMAT exhibited strong activity in acidic environments, maintaining over 85% activity between pH 6.0-8.5 for 60minutes, with peak efficacy at pH 7.0. LfMAT produced 4.2mM SAM from 5mM substrate, indicating reduced product inhibition. Ultimately, using an in vitro multi-enzyme cascade system containing LfMAT, S-adenosylmethionine decarboxylase, and spermidine synthase, we successfully produced 12.9g·L of spermidine. This study establishes a cascade reaction platform, offering a novel approach for the efficient synthesis of spermidine and other polyamines.
Interferon Inhibitors Increase rAAV Production in HEK293 Cells
Recombinant adeno-associated viruses (rAAVs) comprise a promising viral vector for therapeutic gene delivery to treat disease. However, the current manufacturing capability of rAAVs must be improved to meet commercial demand. Previously published omics studies indicate that rAAV production through transient transfection triggers antiviral responses and endoplasmic reticulum stress responses in the host cell. Both responses negatively regulate viral production. We demonstrate that the modulation of the antiviral immune response (by blocking interferon signaling pathways) can effectively lower the production of interferon and enhance viral genome production. The use of interferon inhibitors before transfection can significantly increase rAAV production in HEK293 cells, with up to a 2-fold increase in productivity and up to a 6-fold increase in specific productivity. Compared to the untreated groups, the addition of these small molecules generally reduced viable cell density but increased vector productivity. The positive candidates were BX795 (a TBK inhibitor), TPCA-1 (an IKK2 inhibitor), Cyt387 (a JAK1 inhibitor), and ruxolitinib (another JAK1 inhibitor). These candidates were identified using deep well screening, and reproducible titer improvement was achieved in a 30mL shake flask scale. Additionally, genome titer improvement is feasible and scalable in two different media, but the extent of improvement may vary.
Enhancement of the production of terpenoid and flavonoid secondary metabolites in the ground and aerial parts of licorice composite plant in a hydroponic system
Hairy roots mediated by Agrobacterium rhizogenes can be obtained from the composite plants (plants with hairy roots and untransformed aerial parts) by ex vitro method. Composite plants can produce higher amounts of secondary metabolites by merging hydroponic systems. This provides a stable condition for composite plants, in which various metabolites are produced in different parts. In the present study, composite plants of Glycyrrhiza glabra were produced under ex vitro conditions and transferred into a hydroponic culture medium to produce and extract secondary metabolites. The results showed a 4.8- and 1.8-fold enhancement in the expressions of SQS1 and bAS genes in the roots of the composite plants compared to the control group, respectively. The levels of glycyrrhizin (1.7) and glabridin (3.5) were higher in the leaves of the composite plants compared to the controls. Moreover, higher amounts of glabridin (1.7) were observed in the roots of the composite plants compared to the control group. Investigation of the oxidative enzymes in the composite plants and control group revealed that the plants used more secondary metabolites through Agrobacterium inoculation. The plants needed more antioxidant enzymes to counter the release of oxygen-free radicals in control conditions, but composite plants used secondary metabolites as scavengers. Data revealed that composite plants managed to produce high amounts of various secondary metabolites in a hydroponic system.
A highly efficient mixed strain fermentation strategy to produce 11α-Hydroxyandrost-4-ene-3,17-dione from phytosterols
11α-Hydroxyandrost-4-ene-3,17-dione (11α-OH AD) is an essential steroid hormone drug intermediate that exhibits low biotransformation efficiency. In this study, a mixed-strain fermentation strategy was established for the efficient production of 11α-OH AD from phytosterols (PS). Initially, strains were screened for efficient transformation of AD to produce 11α-OH AD. Subsequently, a dual-strain mixed-culture fermentation technique was established, with Mycolicibacterium neoaurum CICC 21097 ΔksdD (MNR) showing highly effective results. Ultimately, a one-step conversion process for the production of 11α-OH AD was achieved at a molar yield of 76.5 % under optimal conditions using PS as a substrate, the highest reported yield to date. Additionally, studies revealed synergistic metabolic interactions between MNR and Aspergillus ochraceus in the mixed-culture system. These findings provide valuable insights for the industrial production of high-value products using mixed-strain fermentation.
Enhanced methanol-xylose co-utilization strategy in Komagataella phaffii
Bio-manufacturing based on non-food carbon sources is conducive to alleviating the global food crisis and greenhouse effect. However, the mechanism of the utilization of methanol and xylose in Komagataella phaffii based on endogenous metabolic pathways has not been fully explored. In this study, transcriptomics revealed a positive correlation between methanol metabolic efficiency and the transcription level of genes related to xylose metabolism and phosphate metabolism. By providing sufficient phosphate to the strain, the methanol utilization rate of the Komagataella phaffii GA01 strain was improved, and the final biomass reached 7.5g DCW/L. Metabolomics further confirmed that methanol could effectively activate xylose metabolism of the strain, and the consumption rates of methanol and xylose of the Komagataella phaffii GA01 strain could reach 3.87g/L/d and 1.83g/L/d, which were 34% and 357.5% higher than that of the wild-type strain, respectively. This study further promotes the application of methanol and xylose in microbial fermentation.