Microbial Cell Factories最新文献

Background: Echinocandin B (ECB) is a key precursor of the antifungal drug anidulafungin and its biosynthesis occurs via ani gene cluster in Aspergillus nidulans NRRL8112. Strain improvement for industrial ECB production has mainly relied on mutation breeding due to the lack of genetic tools.

Results: Here, a CRISPR-base-editing tool was developed in A. nidulans NRRL8112 for simultaneous inactivation of the nkuA gene and two marker genes, pryoA and riboB, which enabled efficient genetic manipulation. Then, in-vivo plasmid assembly was harnessed for ani gene expression screening, identifying the rate-limiting enzyme AniA and a pathway-specific transcription factor AniJ. Stepwise titer enhancement was achieved by overexpressing aniA and/or aniJ, and ECB production reached 1.5 g/L during 5-L fed-batch fermentation, an increase of ~ 30-fold compared with the parent strain.

Conclusion: This study, for the first time, revealed the regulatory mechanism of ECB biosynthesis and harnessed genetic engineering for the development of an efficient ECB-producing strain.

Background: Uricase is a bio-drug used to reduce urate accumulation in gout disease. Thus, there is a continuous demand for screening soil samples derived from a variety of different sources in order to isolate a strain that possesses a high potential for producing uricase.

Methods: Streptomyces sp. strain NEAE-5 demonstrated a significant capacity for uricase production was identified based on the physiological, morphological and biochemical characteristics, as well as 16S rDNA sequencing analysis. Using a Plackett-Burman statistical design, the impact of eighteen process factors on uricase production by Streptomyces griseorubens strain NEAE-5 was investigated. Using central composite design, the most important variables that had a favourable positive impact on uricase production by Streptomyces griseorubens strain NEAE-5 were further optimized.

Results: It is clear that the morphological and chemotaxonomic features of Streptomyces sp. strain NEAE-5 are typical for the Streptomyces genus. Phylogenetic analysis indicated that Streptomyces sp. strain NEAE-5 belongs to the genus Streptomyces and closely related to Streptomyces griseorubens which it has a 95-96% identity in 16S rDNA gene sequencing. Accordingly, the strain is proposed to be identified as Streptomyces griseorubens strain NEAE-5. The three factors that had the significant positive impacts on uricase production were uric acid, hypoxanthine, and yeast extract. As a result, the best conditions for achieving the highest experimental uricase production by Streptomyces griseorubens strain NEAE-5 after central composite design were (g/L): uric acid 6.96, glycerol 5, hypoxanthine 5.51, MgSO₄.7H₂O 0.1, KNO₃ 2, CaCl₂ 0.5, K₂HPO₄ 0.5, NaCl 0.5, yeast extract 1.08. In addition, the period of incubation is seven days, pH 7.5 and 37 °C with an inoculum size of 2 mL (10⁵ cfu/mL) /100 mL medium.

Conclusions: After optimization, the obtained uricase activity was 120.35 U/mL, indicating that the Streptomyces griseorubens strain NEAE-5 is a potent uricase producer and that the statistical approach used for optimization was appropriate.

Elicitation through abiotic stress, including heavy metals, is a new natural product drug discovery technique. In this research, three compounds 1, 2, and 6, were achieved by triggering zinc and nickel on marine Sphingomonas sp. and Streptomyces sp., which were absent in normal culture. Compound 5 was obtained for the first time from marine bacteria. All compounds showed potent antibacterial activity against Staphylococcus aureus and bactericidal effect at 300 µm, but 6 was more active. The potent compound 6 production was further enhanced through response surface methodology by optimizing the condition consisting of nickel 1 mM ions, 20 mg/L sucrose, 30 mg/L salt and culture time 14 days. Under these conditions, the SM-6 production was enhanced with a yield of 6.3 mg/L, which was absent in the normal culture. Further transcriptome analysis of compound 6 unveiled its antibacterial activity on S. aureus by modulating heat shock protein genes, disrupting protein folding and synthesis, and perturbing cellular redox balance, leading to a comprehensive inhibition of normal bacterial growth. In addition, ADMET has shown that all compounds are safe for cardiac and hepatotoxicity. To determine the anti-bacterial mechanism, all compounds were docked with PBP2a and DNA gyrase enzyme, and TLR-4 protein for predicting vaccine construct, and the best docking score was achieved against PBP2a enzyme with the highest score of -10.2 for compound 6. In-silico cloning was carried out to ensure the expression of proteins generated and were cloned using S.aureus as a host. The simulation studies have shown that both SM-6-PBP2a and TLR-4-PBP2a complex are stable with the system. This study presents a new approach to anti-bacterial drug discovery from microorganisms through heavy metals triggering and enhancing the compound production through response surface methodology.

Background: Since 1982, recombinant insulin has been used as a substitute for pancreatic insulin from animals. However, increasing demand in medical and food industries warrants the development of more efficient production methods. In this study, we aimed to develop a novel and efficient method for insulin production using a yeast secretion system.

Methods: Here, insulin C-peptide was replaced with a hydrophilic fusion partner (HL18) containing an affinity tag for the hypersecretion and easy purification of proinsulin. The HL18 fusion partner was then removed by in vitro processing with the Kex2 endoprotease (Kex2p), and authentic insulin was recovered via affinity chromatography. To improve the insulin functions, molecular chaperones of the host strain were reinforced via the constitutive expression of HAC1.

Results: The developed method was successfully applied for the expression of cow, pig, and chicken insulins in yeast. Moreover, biological activity of recombinant insulins was confirmed by growth stimulation of cell line.

Conclusions: Therefore, replacement of the C-peptide of insulin with the HL18 fusion partner and use of Kex2p for in vitro processing of proinsulin guarantees the economic production of animal insulins in yeast.

Background: Biorefinery using microorganisms to produce biofuels and value-added biochemicals derived from renewable biomass offers a promising alternative to meet our sustainable energy and environmental goals. The ethanologenic strain Zymomonas mobilis is considered as an excellent chassis for constructing microbial cell factories for diverse biochemicals due to its outstanding industrial characteristics in ethanol production, high specific productivity, and Generally Recognized as Safe (GRAS) status. Nonetheless, the restricted substrate range constrains its application.

Results: The truncated ice nucleation protein InaK from Pseudomonas syringae was used as an autotransporter passenger, and α-amylase was fused to the C- terminal of InaK to equip the ethanol-producing bacterium with the capability to ferment renewable biomass. Western blot and flow cytometry analysis confirmed that the amylase was situated on the outer membrane. Whole-cell activity assays demonstrated that the amylase maintained its activity on the cell surface. The recombinant Z. mobilis facilitated the hydrolysis of starch into oligosaccharides and enabled the streamlining of simultaneous saccharification and fermentation (SSF) processes. In a 5% starch medium under SSF, recombinant strains containing P_eno reached a maximum titer of 13.61 ± 0.12 g/L within 48 h. This represents an increase of 111.0% compared to the control strain's titer of titer of 6.45 ± 0.25 g/L.

Conclusions: By fusing the truncated ice nucleation protein InaK with α-amylase, we achieved efficient expression and surface display of the enzyme on Z. mobilis. This fusion protein exhibited remarkable enzymatic activity. Its presence enabled a cost-effective bioproduction process using starch as the sole carbon source, and it significantly reduced the required cycle time for SSF. This study not only provides an excellent Z. mobilis chassis for sustainable bioproduction from starch but also highlights the potential of Z. mobilis to function as an effective cellular factory for producing high-value products from renewable biomass.

Background: Acetate is an important chemical feedstock widely applied in the food, chemical and textile industries. It is now mainly produced from petrochemical materials through chemical processes. Conversion of lignocellulose biomass to acetate by biotechnological pathways is both environmentally beneficial and cost-effective. However, acetate production from carbohydrate in lignocellulose hydrolysate via glycolytic pathways involving pyruvate decarboxylation often suffers from the carbon loss and results in low acetate yield.

Results: Escherichia coli BL21 (DE3) was confirmed to have high tolerance to acetate in this work. Thus, it was selected from seven laboratory E. coli strains for acetate production from lignocellulose hydrolysate. The byproduct-producing genes frdA, ldhA, and adhE in E. coli BL21 (DE3) were firstly knocked out to decrease the generation of succinate, lactate, and ethanol. Then, the genes pfkA and edd were also deleted and bifunctional phosphoketolase and fructose-1,6-bisphosphatase were overexpressed to construct an EP-bifido pathway in E. coli BL21 (DE3) to increase the generation of acetate from glucose. The obtained strain E. coli 5K/pFF can produce 22.89 g/L acetate from 37.5 g/L glucose with a yield of 0.61 g/g glucose. Finally, the ptsG gene in E. coli 5K/pFF was also deleted to make the engineered strain E. coli 6K/pFF to simultaneously utilize glucose and xylose in lignocellulosic hydrolysates. E. coli 6K/pFF can produce 20.09 g/L acetate from corn stover hydrolysate with a yield of 0.52 g/g sugar.

Conclusion: The results presented here provide a promising alternative for acetate production with low cost substrate. Besides acetate production, other biotechnological processes might also be developed for other acetyl-CoA derivatives production with lignocellulose hydrolysate through further metabolic engineering of E. coli 6K/pFF.

Background: Microalgae have emerged as sustainable alternatives to fossil fuels and high-value petrochemicals. Despite the commercial potential of microalgae, their low biomass productivity is a significant limiting factor for large-scale production. In the photoautotrophic cultivation of microalgae, achievable cell density levels depend on the light transmittance of the production system, which can significantly decrease the photosynthetic rate and biomass production. In contrast, the mixotrophic cultivation of microalgae using heterotrophic carbon sources enables high-density cultivation, which significantly enhances biomass productivity. The identification of optimal production conditions is crucial for improving biomass productivity; however, it is typically time- and resource-consuming. To overcome this problem, high-throughput screening (HTS) system presents a practical approach to maximize biomass and lipid production and enhance the industrial applicability of microalgae.

Results: In this study, we proposed a two-step HTS assay that allows effective screening of heterotrophic conditions compatible with new microalgal isolates. To confirm the effectiveness of the HTS assay, three microalgal isolates with distinctive morphological and genetic traits were selected. Suitable cultivation conditions, including various heterotrophic carbon sources, substrate concentrations, and temperatures, were investigated using a two-step HTS assay. The optimized conditions were validated at the flask scale, which confirmed a significant enhancement in the biomass and lipid productivity of each isolate. Moreover, the two-step HTS assay notably enhanced economic and temporal efficiency compared to conventional flask-based optimization.

Conclusions: These results suggest that our two-step HTS assay is an efficient strategy for investigating and optimizing microalgal culture conditions to maximize biomass and lipid productivity. This approach has the potential to enhance the industrial applicability of microalgae and facilitate the seamless transition from laboratory to field applications.

Zeaxanthin, a vital dietary carotenoid, is naturally synthesized by plants, microalgae, and certain microorganisms. Large-scale zeaxanthin production can be achieved through plant extraction, chemical synthesis, or microbial fermentation. The environmental and health implications of the first two methods have made microbial fermentation an appealing alternative for natural zeaxanthin production despite the challenges in scaling up the bioprocess. An intermediate between β-carotene and zeaxanthin, β-cryptoxanthin, is found only in specific fruits and vegetables and has several important functions for human health. The low concentration of β-cryptoxanthin in these sources results in low extraction yields, making biotechnological production a promising alternative for achieving higher yields. Currently, there is no industrially relevant microbial fermentation process for β-cryptoxanthin production, primarily due to the lack of identified enzymes that specifically convert β-carotene to β-cryptoxanthin without further conversion to zeaxanthin. In this study, we used genetic engineering to leverage the oleaginous yeast Yarrowia lipolytica as a bio-factory for zeaxanthin and β-cryptoxanthin production. We screened 22 β-carotene hydroxylases and identified eight novel enzymes with β-carotene hydroxylating activity: six producing zeaxanthin and two producing only β-cryptoxanthin. By introducing the β-carotene hydroxylase from the bacterium Chondromyces crocatus (CcBCH), a β-cryptoxanthin titer of 24 ± 6 mg/L was achieved, representing the highest reported titer of sole β-cryptoxanthin in Y. lipolytica to date. By targeting zeaxanthin-producing β-carotene hydroxylase to the endoplasmic reticulum and peroxisomes, we increased the production of zeaxanthin by 54% and 66%, respectively, compared to untargeted enzyme. The highest zeaxanthin titer of 412 ± 34 mg/L was achieved by targeting β-carotene hydroxylases to peroxisomes. In addition, by constructing multienzyme scaffold-free complexes with short peptide tags RIDD and RIAD, we observed a 39% increase in the zeaxanthin titer and a 28% increase in the conversion rate compared to the strain expressing unmodified enzyme. The zeaxanthin titers obtained in this study are not the highest reported; however, our goal was to demonstrate that specific approaches can enhance both titer and conversion rate, rather than to achieve the maximum titer. These findings underscore the potential of Y. lipolytica as a promising platform for carotenoid production and provide a foundation for future research, where further optimization is required to maximize production.

Background: Limited research has been conducted on energy fluctuation during the transition state, despite the critical role of energy supply in microbial physiological metabolism.

Results: This study aimed to investigate the regulatory function of transition state transcription factor AbrB on energy metabolism in Bacillus licheniformis WX-02. Firstly, the deletion of abrB was found to prolong the cell generation time, significantly reducing the intercellular ATP concentration and NADH/NAD⁺ ratio at the early stage. Subsequently, various target genes and transcription factors regulated by AbrB were identified through in vitro verification assays. Specifically, AbrB was shown to modulate energy metabolism by directly regulating the expression of genes pyk and pgk in substrate-level phosphorylation, as well as genes narK and narGHIJ associated with nitrate respiration. In terms of oxidative phosphorylation, AbrB not only directly regulated ATP generation genes, including cyd, atpB, hmp, ndh, qoxA and sdhC, but also influenced the expression of NAD-dependent enzymes and intracellular NADH/NAD⁺ ratio. Additionally, AbrB positively affected the expression of transcription factors CcpN, Fnr, Rex, and ResD involved in energy supply, while negatively affected the regulator CcpA. Overall, this study found that AbrB positively regulates both substrate-level phosphorylation and oxidative phosphorylation, while negatively regulating nitrate respiration.

Conclusions: This study proposes a comprehensive regulatory network of AbrB on energy metabolism in Bacillus, expanding the understanding of regulatory mechanisms of AbrB and elucidating energy fluctuations during the transition state.

Recently, methane has been considered a next-generation carbon feedstock due to its abundance and it is main component of shale gas and biogas. Methylomonas sp. DH-1 has been evaluated as a promising industrial bio-catalyst candidate. Succinate is considered one of the top building block chemicals in the agricultural, food, and pharmaceutical industries. In this study, succinate production by Methylomonas sp. DH-1 was improved by combining adaptive laboratory evolution (ALE) technology with genetic engineering in the chromosome of Methylomonas sp. DH-1, such as deletion of bypass pathway genes (succinate dehydrogenase and succinate semialdehyde dehydrogenase) or overexpression of genes related with succinate production (citrate synthase, pyruvate carboxylase and phosphoenolpyruvate carboxylase). Through ALE, the maximum consumption rate of substrate gases (methane and oxygen) and the duration maintaining high substrate gas consumption rates was enhanced compared to those of the parental strain. Based on the improved methane consumption, cell growth (OD₆₀₀) increased more than twice, and the succinate titer increased by ~ 48% from 218 to 323 mg/L. To prevent unwanted succinate consumption, the succinate semialdehyde dehydrogenase gene was deleted from the genome. The first enzyme of TCA cycle (citrate synthase) was overexpressed. Pyruvate carboxylase and phosphoenolpyruvate carboxylase, which produce oxaloacetate, a substrate for citrate synthase, were also overproduced by a newly identified strong promoter. The new strong promoter was screened from RNA sequencing data. When these modifications were combined in one strain, the maximum titer (702 mg/L) was successfully improved by more than three times. This study demonstrates that successful enhancement of succinic acid production can be achieved in methanotrophs through additional genetic engineering following adaptive laboratory evolution.