Deletion of genomic islands in the Pseudomonas putida KT2440 genome can create an optimal chassis for synthetic biology applications.

BACKGROUND: Genome streamlining is a feasible strategy for constructing an optimum microbial chassis for synthetic biology applications. Genomic islands (GIs) are usually regarded as foreign DNA sequences, which can be obtained by horizontal gene transfer among microorganisms. A model strain Pseudomonas putida KT2440 has broad applications in biocatalysis, biotransformation and biodegradation. RESULTS: In this study, the identified GIs in P. putida KT2440 accounting for 4.12% of the total genome size were deleted to generate a series of genome-reduced strains. The mutant KTU-U13 with the largest deletion was advantageous over the original strain KTU in several physiological characteristics evaluated. The mutant KTU-U13 showed high plasmid transformation efficiency and heterologous protein expression capacity compared with the original strain KTU. The metabolic phenotype analysis showed that the types of carbon sources utilized by the mutant KTU-U13 and the utilization capabilities for certain carbon sources were increased greatly. The polyhydroxyalkanoate (PHA) yield and cell dry weight of the mutant KTU-U13 were improved significantly compared with the original strain KTU. The chromosomal integration efficiencies for the γ-hexachlorocyclohexane (γ-HCH) and 1,2,3-trichloropropane (TCP) biodegradation pathways were improved greatly when using the mutant KTU-U13 as the recipient cell and enhanced degradation of γ-HCH and TCP by the mutant KTU-U13 was also observed. The mutant KTU-U13 was able to stably express a plasmid-borne zeaxanthin biosynthetic pathway, suggesting the excellent genetic stability of the mutant. CONCLUSIONS: These desirable traits make the GIs-deleted mutant KTU-U13 an optimum chassis for synthetic biology applications. The present study suggests that the systematic deletion of GIs in bacteria may be a useful approach for generating an optimal chassis for the construction of microbial cell factories.

Enhancing the thermotolerance and erythritol production of Yarrowia lipolytica by introducing heat-resistant devices.

Yarrowia lipolytica has been widely used in the food biotech-related industry, where it plays the host's role in producing erythritol. Nevertheless, a temperature of about 28°C-30°C has been estimated as the yeast's optimal growth temperature, leading to the consumption of a considerable quantity of cooling water, especially in summer, which is obligatory for fermentation. Herein is described a method for improving the thermotolerance and erythritol production efficiency at high temperatures of Y. lipolytica. Through screening and testing different heat resistant devices, eight refactored engineered strains showed better growth at higher temperature and the antioxidant properties of the eight engineered strains were also improved. In addition, the erythritol titer, yield and productivity of the strain FOS11-Ctt1 represented the best among the eight strains, reaching at 39.25 g/L, 0.348 g/g glucose, and 0.55 g/L/h respectively, which were increased by 156%, 86% and 161% compared with the control strain, respectively. This study provides insight into an effective heat-resistant device that could enhance the thermotolerance and erythritol production of Y. lipolytica, which might be considered a valued scientific reference for other resistant strains' construction.

Expanding sugar alcohol industry: Microbial production of sugar alcohols and associated chemocatalytic derivatives.

Sugar alcohols are polyols that are widely employed in the production of chemicals, pharmaceuticals, and food products. Chemical synthesis of polyols, however, is complex and necessitates the use of hazardous compounds. Therefore, the use of microbes to produce polyols has been proposed as an alternative to traditional synthesis strategies. Many biotechnological approaches have been described to enhancing sugar alcohols production and microbe-mediated sugar alcohol production has the potential to benefit from the availability of inexpensive substrate inputs. Among of them, microbe-mediated erythritol production has been implemented in an industrial scale, but microbial growth and substrate conversion rates are often limited by harsh environmental conditions. In this review, we focused on xylitol, mannitol, sorbitol, and erythritol, the four representative sugar alcohols. The main metabolic engineering strategies, such as regulation of key genes and cofactor balancing, for improving the production of these sugar alcohols were reviewed. The feasible strategies to enhance the stress tolerance of chassis cells, especially thermotolerance, were also summarized. Different low-cost substrates like glycerol, molasses, cellulose hydrolysate, and CO2 employed for producing these sugar alcohols were presented. Given the value of polyols as precursor platform chemicals that can be leveraged to produce a diverse array of chemical products, we not only discuss the challenges encountered in the above parts, but also envisioned the development of their derivatives for broadening the application of sugar alcohols.

[Advances in metabolic engineering of non-conventional yeasts].

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.

Efficacy of Various Preservatives on Extending Shelf Life of Vacuum-Packaged Raw Pork during 4°C Storage.

Uncontrolled bacterial growth and metabolic activities are responsible for the short shelf life of raw pork. Culture-independent analysis by 16S ribosome cDNA could reveal viable bacteria in raw pork. This study investigated microbial growth and volatile organic compounds of raw pork supplemented with various preservatives. Vacuum-packaged raw pork was stored at 4°C, after soaking in solutions of potassium sorbate, ε-poly-l-lysine, kojic acid (KA), or sodium diacetate, individually. Spoilage of raw pork was monitored by determining pH and total volatile basic nitrogen, whereas bacterial growth was determined by culture-dependent and culture-independent analyses. Data indicated that all the preservatives were able to inhibit bacterial growth and extend the shelf life of pork. High-throughput sequencing of 16S ribosome cDNA indicated that Pseudomonas was inhibited under vacuum conditions, whereas facultative anaerobes ( Acinetobacter, Photobacterium, Brochothrix, and Myroides) were the most active genera in the spoiled pork. Photobacterium was further inhibited by each preservative. The inhibition of Acinetobacter, Photobacterium, and Myroides could be responsible for the extended shelf life of vacuum-packaged pork; they were effectively inhibited by KA, which also induced the longest shelf life. Moreover, 19 types of volatile organic compounds were detected. 3-Methylbutanol, 3-methylbutanol acetate, 2-butanone, toluene, benzeneacetaldehyde, dimethyl trisulfide, and acetoin were associated with spoilage. Furthermore, KA is a potential preservative in raw pork; because no phenol was detectable within 35 days, excessive intake of phenol induced by preservatives was avoided.

An engineered Pseudomonas putida can simultaneously degrade organophosphates, pyrethroids and carbamates.

Agricultural soils are often polluted with a variety of pesticides. Unfortunately, natural microorganisms lack the capacity to simultaneously degrade different types of pesticides. Currently, synthetic biology provides powerful approaches to create versatile degraders. In this work, a biosafety strain Pseudomonas putida KT2440 was engineered for simultaneous degradation of organophosphates, pyrethroids, and carbamates, enhanced oxygen-sequestering capability, and real-time monitoring by targeted insertion of four pesticide-degrading genes, vgb, and gfp into the chromosome using a scarless genome-editing method. The resulting recombinant strain, designated as P. putida KTUe, could completely degrade 50mg/L methyl parathion, chlorpyrifos, fenpropathrin, cypermethrin, carbofuran and carbaryl within 30h when incubated in M9 minimal medium supplemented with 20g/L glucose. In soil remediation studies, all the tested six pesticides (50mg/kg soil each) were completely removed in soils inoculated with P. putida KTUe within 15days. Moreover, Vitreoscilla hemoglobin (VHb)-expressing P. putida KTUe grew faster than P. putida KTUd without VHb expression under oxygen-limited conditions, suggesting that VHb may enhance the capability of this recombinant strain to sequester oxygen. Furthermore, the green fluorescence was observed on the P. putida KTUe cells, suggesting that this green fluorescent protein (GFP)-marked strain may be tracked by fluorescence during bioremediation. Therefore, this recombinant strain may serve as a promising candidate for in situ bioremediation of soil contaminated with multiple pesticides. This work not only underscores the value of P. putida KT2440 as an ideal host for bioremediation but also highlights the power of synthetic biology for expanding the degradation capability of natural degraders.

High-Throughput Sequencing of Viable Microbial Communities in Raw Pork Subjected to a Fast Cooling Process.

This study aimed to investigate the effect of the fast cooling process on the microbiological community in chilled fresh pork during storage. We established a culture-independent method to study viable microbes in raw pork. Tray-packaged fresh pork and chilled fresh pork were completely spoiled after 18 and 49 d in aseptic bags at 4 °C, respectively. 16S/18S ribosomal RNAs were reverse transcribed to cDNA to characterize the activity of viable bacteria/fungi in the 2 types of pork. Both cDNA and total DNA were analyzed by high-throughput sequencing, which revealed that viable Bacteroides sp. were the most active genus in rotten pork, although viable Myroides sp. and Pseudomonas sp. were also active. Moreover, viable fungi were only detected in chilled fresh pork. The sequencing results revealed that the fast cooling process could suppress the growth of microbes present initially in the raw meat to extend its shelf life. Our results also suggested that fungi associated with pork spoilage could not grow well in aseptic tray-packaged conditions.

Elucidation of major contributors involved in nitrogen removal and transcription level of nitrogen-cycling genes in activated sludge from WWTPs.

We investigated nitrogen-cycle bacterial communities in activated sludge from 8 municipal wastewater treatment plants (WWTPs). Redundancy analyses (RDA) showed that temperature was the most significant driving force in shaping microbial community structure, followed by influent NH4+ and total nitrogen (TN). The diversity of ammonia oxidizing and nitrite reducing bacteria were investigated by the construction of amoA, nirS and nirK gene clone libraries. Phylogenetic analysis indicated that Thauera and Mesorhizobium were the predominant nitrite reducing bacteria, and Nitrosomonas was the only detected ammonia oxidizing bacteria in all samples. Quantification of transcription level of nirS and nirK genes indicated that nirS-type nitrite reducing bacteria played the dominant roles in nitrite reduction process. Transcription level of nirS gene positively correlated with influent NH4+ and TN significantly, whereas inversely linked with hydraulic retention time. Temperature had a strong positive correlation to transcription level of amoA gene. Overall, this study deepened our understanding of the major types of ammonia oxidizing and nitrite reducing bacteria in activated sludge of municipal WWTPs. The relationship between transcription level of nitrogen-cycle genes and operational or environmental variables of WWTPs revealed in this work could provide guidance for optimization of operating parameters and improving the performance of nitrogen removal.