medium optimization
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2022 ◽  
Author(s):  
Amir Akhgari ◽  
Bikash Baral ◽  
Arina Koroleva ◽  
Vilja Siitonen ◽  
David P Fewer ◽  
...  

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l-1 in a second round of mutant selection. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g-1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.


2021 ◽  
Vol 111 ◽  
pp. 139-146
Author(s):  
Xiaoying Zhang ◽  
Qingbin Wu ◽  
Xiaoyuan Zhang ◽  
Zhongyuan Lv ◽  
Xiaoting Mo ◽  
...  

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Umesh Luthra ◽  
Prabhakar Babu ◽  
Remya R.R. ◽  
Angeline Julius ◽  
Yogesh Patel ◽  
...  

Purpose β-Carotene is the most appropriate and significant precursor of vitamin A. Synthetic carotene supplements have been known to pose a threat to human health, making natural sources such as the indefensible choice for the production and extraction of carotene. Design/methodology/approach This study considers Blakeslea trispora, a filamentous fungus, as a source of production of carotenoids by fermentation and wet and dry mycelium were used to analyse and obtain better extraction results. Findings In this study, natural oils such as soy oil and cottonseed oil were incorporated into fermentation media to increase the production of carotene. For the optimization process, Plackett–Burman and one-factor-at-a-time (OVAT) models were identified as being of great value. Originality/value OVAT was carried out for corn starch because it plays a major role in the production of carotene and the corn starch at 30 g/L concentration has shown the maximum activity of 3.48 mg/gm. After optimizing process variables, submerged fermentation was eventually carried out under highly controlled media conditions. The resulting product was quantified using UV spectroscopy and extraction of carotene has been observed in the presence of various solvents. Among a range of solvents used, the methylene Di chloride produced-carotene at 86% recovery at a significantly lower temperature of 35°C.


Author(s):  
Zhiying Fan ◽  
Nian Tong ◽  
Zhoukang Zhuang ◽  
Cheng Ma ◽  
Junying Ma ◽  
...  

Tuberculosis (TB) and its emerged drug resistance exert huge threats on the global health, therefore development of novel anti-TB antibiotics is very essential. Ilamycin-E1/E2 is a pair of cycloheptapeptide enantiomers obtained from a marine-derived Streptomyces atratus SCSIO ZH16-ΔilaR mutant, and become promising anti-TB lead compounds due to their significant anti-TB activities, but their low titer hampered the further clinical development. In this work, the statistical Plackett-Burman design (PBD) model was applied to screen out bacterial peptone as the only significant but negative factor affecting the ilamycin-E1/E2 production. Subsequent single factor optimization revealed that replacement of bacterial peptone with malt extract eliminated the accumulation of porphyrin-type competitive byproduct, and the titer of ilamycin-E1/E2 in shaking flasks was improved from original 13.6±0.8 to 142.7±5.7 mg/L for about 10.5 folds. Furthermore, a pH coordinated feeding strategy was first adopted in scaled-up production of ilamycin-E1/E2. The obtained titer of ilamycin-E1/E2 in 30L was 169.8±2.5 mg/L, while in 300L fermentor was only 131.5±7.5 mg/L due to the unsynchronization of feeding response and pH change. Therefore, the continuous pulse feeding strategy was further applied in 300L fermentor and finally achieved 415.7±29.2 mg/L ilamycin-E1/E2, which represented about 30.5 folds improvement at last. Our work provided the solid basis to achieve sufficient ilamycin-E1/E2 lead compounds and support their potential anti-TB drug development.


2021 ◽  
Vol 189 ◽  
pp. 112852
Author(s):  
Xue-Juan Pu ◽  
Qian-Yi Hu ◽  
Su-Su Li ◽  
Guo-Hong Li ◽  
Pei-Ji Zhao

2021 ◽  
Author(s):  
Bingyu Yan ◽  
Wen Gao ◽  
Li Tian ◽  
Shuai Wang ◽  
Huijun Dong

Abstract Objective To enhance the production of A40926 by implementing a strategy of the combination of genetically engineered strain construction and medium optimization. Results The engineered strain of Nonomuraea gerenzanensis presented an increment of 30.6 percent in A40926 production compared with that of the parent strain. Subsequently, an assembling medium, which was defined as M9 medium and mainly comprised glucose, maltodextrin, soybean meal, peptone, L-valine, and other inorganic salts, was determined as the optimal medium among the tested nine media. The optimum concentration of medium components was glucose 10 g/l, maltodextrin 37.9 g/l, soybean meal 34.5 g/l, peptone 30.0 g/l, and L-valine 4.3 g/l, respectively. The optimized medium was verified experimentally, and A40926 yield increased significantly from 257 mg/l to 332 mg/l, as compared to the non-optimized medium. The strategy brought a significant increase of A40926 yield by 65.2 percent. Conclusions The engineered mutant with the genetic attributes of the co-expression of the dbv3 and dbv20 genes and the deletion of the dbv23 gene could obviously enhance the production of A40926. In addition, the optimization of medium was an effective and essential tool for the improvement of the secondary metabolites in Actinomyces.


LWT ◽  
2021 ◽  
pp. 111812
Author(s):  
Yu Lu ◽  
Xiangjin Cheng ◽  
Huanhuan Deng ◽  
Shouwen Chen ◽  
Zhixia Ji

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