Improved Neomycin Sulfate Potency in Streptomyces fradiae Using Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis and Fermentation Medium Optimization

To improve the screening efficiency of high-yield neomycin sulfate (NM) Streptomyces fradiae strains after mutagenesis, a high-throughput screening method using streptomycin resistance prescreening (8 μg/mL) and a 24-deep well plates/microplate reader (trypan blue spectrophotometry) rescreening strategy was developed. Using this approach, we identified a high-producing NM mutant strain, Sf6-2, via six rounds of atmospheric and room temperature plasma (ARTP) mutagenesis and screening. The mutant displayed a NM potency of 7780 ± 110 U/mL and remarkably stable genetic properties over six generations. Furthermore, the key components (soluble starch, peptone, and (NH4)2SO4) affecting NM potency in fermentation medium were selected using Plackett-Burman and optimized by Box-Behnken designs. Finally, the NM potency of Sf6-2 was increased to 10,849 ± 141 U/mL at the optimal concentration of each factor (73.98 g/L, 9.23 g/L, and 5.99 g/L, respectively), and it exhibited about a 40% and 100% enhancement when compared with before optimization conditions and the wild-type strain, respectively. In this study, we provide a new S. fradiae NM production strategy and generate valuable insights for the breeding and screening of other microorganisms.

A Novel Approach to Develop Lager Yeast with Higher NADH Availability to Improve the Flavor Stability of Industrial Beer

Flavor stability is important for beer quality and extensive efforts have been undertaken to improve this. In our previous work, we proved a concept whereby metabolic engineering lager yeast with increased cellular nicotinamide adenine dinucleotide hydride (NADH) availability could enhance the flavor stability of beer. However, the method for breeding non-genetically modified strains with higher NADH levels remains unsolved. In the current study, we reported a novel approach to develop such strains based on atmospheric and room temperature plasma (ARTP) mutagenesis coupled with 2,4-dinitrophenol (DNP) selection. As a result, we obtained a serial of strains with higher NADH levels as well as improved flavor stability. For screening an optimal strain with industrial application potential, we examined the other fermentation characteristics of the mutants and ultimately obtained the optimal strain, YDR-63. The overall fermentation performance of the strain YDR-63 in pilot-scale fermentation was similar to that of the parental strain YJ-002, but the acetaldehyde production was decreased by 53.7% and the resistance staling value of beer was improved by 99.8%. The forced beer aging assay further demonstrated that the favor stability was indeed improved as the contents of 5-hydroxymethylfurfural in YDR-63 was less than that in YJ-002 and the sensory notes of staling was weaker in YDR-63. We also employed this novel approach to another industrial strain, M14, and succeeded in improving its flavor stability. All the findings demonstrated the efficiency and versatility of this new approach in developing strains with improved flavor stability for the beer industry.

Microfluidic Screening And Genomic Mutation Identification For Enhancing Cellulase Production in Pichia Pastoris

BackgroundPichia pastoris is a widely used host organism for heterologous production of industrial enzymes and other proteins, such as cellulase applied in various industries. By developing rational and irrational engineering strategies, great progress has been achieved in improving protein expression in P. pastoris. However, due to unknown genomic impact factors, the potential of the P. pastoris expression system has not been fully explored. Recently, whole-cell directed evolution, employing iterative rounds of genome-wide diversity generation and high throughput screening (HTS), is considered as a promising strategy in strain improvement at the genome level.ResultsIn this study, whole-cell directed evolution of P. pastoris, employing atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic high-throughput screening (HTS), was developed to improve heterogenous cellulase production. The droplet-based microfluidic HTS platform based on a cellulase-catalyzed reaction of releasing fluorescence was established to be suitable for methanol-grown P. pastoris. The validation experiment showed a positive sorting efficiency of 94.4% at a sorting rate of 300 drops per second. Through five rounds of iterative ARTP mutagenesis and microfluidic screening, cellulase production capacities of mutants in each round were gradually enhanced. Among them, the best mutant strain R5-2 exhibited the cellulase activity of 11,110 ± 523 U/L, an about 2-fold increase compared to the starting strain. Whole-genome resequencing analysis further uncovered three accumulated genomic alterations in coding region during iterative ARTP mutagenesis and screening. Intriguingly, the point mutation Rsc1V22G was observed in all the top-performing producers selected from each round, and gene deletion analysis confirmed that Rsc1, a component of the RSC chromatin remodeling complex, might play an important role in cellulase production. ConclusionsWe established a droplet-based microfluidic HTS system, thereby facilitating whole-cell directed evolution of P. pastoris for enhancing cellulase production, and meanwhile identified genomic alterations by whole-genome resequencing and analyzed. Our approaches and findings would provide guides to accelerate whole-cell directed evolution of host strains and enzymes of high industrial interest.

Metabolic Analysis of Schizochytrium Mutants With High DHA Content Achieved With ARTP Mutagenesis Combined With Iodoacetic Acid and Dehydroepiandrosterone Screening

High DHA production cost caused by low DHA titer and productivity of the current Schizochytrium strains is a bottleneck for its application in competition with traditional fish-oil based approach. In this study, atmospheric and room-temperature plasma with iodoacetic acid and dehydroepiandrosterone screening led to three mutants, 6–8, 6–16 and 6–23 all with increased growth and DHA accumulations. A LC/MS metabolomic analysis revealed the increased metabolism in PPP and EMP as well as the decreased TCA cycle might be relevant to the increased growth and DHA biosynthesis in the mutants. Finally, the mutant 6–23, which achieved the highest growth and DHA accumulation among all mutants, was evaluated in a 5 L fermentor. The results showed that the DHA concentration and productivity in mutant 6–23 were 41.4 g/L and 430.7 mg/L/h in fermentation for 96 h, respectively, which is the highest reported so far in literature. The study provides a novel strain improvement strategy for DHA-producing Schizochytrium.

ARTP Mutagenesis of Schizochytrium sp. PKU#Mn4 and Clethodim-Based Mutant Screening for Enhanced Docosahexaenoic Acid Accumulation

Schizochytrium species are one of the best oleaginous thraustochytrids for high-yield production of docosahexaenoic acid (DHA, 22:6). However, the DHA yields from most wild-type (WT) strains of Schizochytrium are unsatisfactory for large-scale production. In this study, we applied the atmospheric and room-temperature plasma (ARTP) tool to obtain the mutant library of a previously isolated strain of Schizochytrium (i.e., PKU#Mn4). Two rounds of ARTP mutagenesis coupled with the acetyl-CoA carboxylase (ACCase) inhibitor (clethodim)-based screening yielded the mutant A78 that not only displayed better growth, glucose uptake and ACCase activity, but also increased (54.1%) DHA content than that of the WT strain. Subsequent optimization of medium components and supplementation improved the DHA content by 75.5% and 37.2%, respectively, compared with that of mutant A78 cultivated in the unoptimized medium. Interestingly, the ACCase activity of mutant A78 in a medium supplemented with biotin, citric acid or sodium citrate was significantly greater than that in a medium without supplementation. This study provides an effective bioengineering approach for improving the DHA accumulation in oleaginous microbes.

ARTP Mutagenesis to Improve Mycelial Polysaccharide Production of Grifola frondosa Using a Mixture of Wheat Bran and Rice Bran as Substrate

Mycelial polysaccharides from Grifola frondosa have shown potential for the prevention of chronic diseases. Atmospheric and room temperature plasma (ARTP) technology was used to enhance the ability of G. frondosa to efficiently utilize a mixture of rice bran and wheat bran in the production of mycelial polysaccharides. The ARTP-mutant G. frondosa GFA2 had an improved growth rate of 6.0 mm/d and polysaccharide yield of 2.65 g/L and showed stable genetic characteristics. Uniform design experiments showed that polysaccharide yield could be increased to 5.90 g/L using the optimized conditions of 10.0 g/L rice bran and 110.0 g/L wheat bran while omitting KH2PO4 and MgSO4·7H2O. Gas chromatography demonstrated that GFA2 polysaccharides were composed of the monosaccharides rhamnose, arabinose, fucose, xylose, mannose, glucose, and galactose. This study provides an effective strategy for improving polysaccharide production in edible fungi while proposing the added-value utilization of rice and wheat brans.

Combination of Atmospheric and Room Temperature Plasma (ARTP) mutagenesis, Genome shuffling and Dimethyl sulfoxide (DMSO) feeding to improve FK506 production in Streptomyces tsukubaensis

FK506 is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces tsukubaensis . However, the production capacity of the strain is very low. To improve production, atmospheric and room temperature plasma (ARTP)mutagenesis was adopted to get the initial strains used in genome shuffling (GS). After three rounds of GS, S. tsukubaensis R3-C4 was the most productive strain, resultingin a FK506 concentration of 335 μg/mL, 2.6 times than that of the originalwild-type strain. Moreover, exogenous DMSO 4 % (v/v) addition could induce efflux of FK506 and increased FK506 production by 27.9% to 429 μg/mL . Finally, analyses of the differences in morphology, fermentation characteristics and specific gene expression levels between S. tsukubaensis R3-C4 and the wild-type strain revealed that R3-C4 strain: has hampered spore differentiation , thicker mycelia and more red pigment, which are likely related to the downregulation of bldD and cdgB expression. In addition, the expression levels of fkbO , fkbP , dahp , pccB and prpE all showed up-regulation at diverse degrees compared to the wild-type S. tsukubaensis . Overall, these results show that a combined approach involving classical random mutation and exogenous feeding canbe applied to increase FK506 biosynthesis andmay be applied also tothe improvement of other important secondary metabolites.