Establishment of low-cost production platforms of polyhydroxyalkanoate bioplastics from Halomonas cupida J9.

Microbial production of polyhydroxyalkanoate (PHA) is greatly restricted by high production cost arising from high-temperature sterilization and expensive carbon sources. In this study, a low-cost PHA production platform was established from Halomonas cupida J9. First, a marker-less genome-editing system was developed in H. cupida J9. Subsequently, H. cupida J9 was engineered to efficiently utilize xylose for PHA biosynthesis by introducing a new xylose metabolism module and blocking xylonate production. The engineered strain J9UΔxylD-P8xylA has the highest PHA yield (2.81 g/L) obtained by Halomonas with xylose as the sole carbon source so far. This is the first report on the production of short- and medium-chain-length (SCL-co-MCL) PHA from xylose by Halomonas. Interestingly, J9UΔxylD-P8xylA was capable of efficiently utilizing glucose and xylose as co-carbon sources for PHA production. Furthermore, fed-batch fermentation of J9UΔxylD-P8xylA coupled to a glucose/xylose co-feeding strategy reached up to 12.57 g/L PHA in a 5-L bioreactor under open and unsterile condition. Utilization of corn straw hydrolysate as the carbon source by J9UΔxylD-P8xylA reached 7.0 g/L cell dry weight (CDW) and 2.45 g/L PHA in an open fermentation. In summary, unsterile production in combination with inexpensive feedstock highlights the potential of the engineered strain for the low-cost production of PHA from lignocellulose-rich agriculture waste.

Metabolic engineering of genome-streamlined strain Pseudomonas putida KTU-U27 for medium-chain-length polyhydroxyalkanoate production from xylose and cellobiose.

Bio-based plastics polyhydroxyalkanoates (PHAs) are considered as a good substitutive to traditional fossil-based plastics because PHAs outcompete chemical plastics in several important properties, such as biodegradability, biocompatibility, and renewability. However, the industrial production of PHA (especially medium-chain-length PHA, mcl-PHA) is greatly restricted by the cost of carbon sources. Currently, xylose and cellobiose derived from lignocellulose are potential substrates for mcl-PHA production. In this study, Pseudomonas putida KTU-U27, a genome-streamlined strain derived from a mcl-PHA producer P. putida KT2440, was used as the optimal chassis for the construction of microbial cell factories with the capacity to efficiently produce mcl-PHA from xylose and cellobiose by introducing the xylose and cellobiose metabolism modules and enhancing the transport of xylose and cellobiose. The lag phases of the xylose- and cellobiose-grown engineered strains were almost completely eliminated and the xylose- and cellobiose-utilizing performance was greatly improved via adaptive laboratory evolution. In shake-flask fermentation, the engineered strain 27A-P13-xylABE-Ptac-tt and 27A-P13-bglC-P13-gts had a mcl-PHA content of 41.67 wt% and 45.18 wt%, respectively, and were able to efficiently utilize xylose or cellobiose as the sole carbon source for cell growth. Herein, microbial production of mcl-PHA using xylose as the sole carbon source has been demonstrated for the first time. Meanwhile, the highest yield of mcl-PHA produced from cellobiose has been obtained in this study. Interestingly, the engineered strains derived from genome-reduced P. putida strains showed higher xylose- and cellobiose-utilizing performance and higher PHA yield than those derived from P. putida KT2440. This study highlights enormous potential of the engineered strains as promising platforms for low-cost production of mcl-PHA from xylose- and cellobiose-rich substrates.

Creating an efficient 1,2-dichloroethane-mineralizing bacterium by a combination of pathway engineering and promoter engineering.

Currently, 1,2-dichloroethane (DCA) is frequently detected in groundwater and has been listed as a potential human carcinogen by the U.S. EPA. Owing to its toxicity and recalcitrant nature, inefficient DCA mineralization has become a bottleneck of DCA bioremediation. In this study, the first engineered DCA-mineralizing strain KTU-P8DCA was constructed by functional assembly of DCA degradation pathway and enhancing pathway expression with a strong promoter P8 in the biosafety strain Pseudomonas putida KT2440. Strain KTU-P8DCA can metabolize DCA to produce CO2 and utilize DCA as the sole carbon source for cell growth by quantifying 13C stable isotope ratios in collected CO2 and in lyophilized cells. Strain KTU-P8DCA exhibited superior tolerance to high concentrations of DCA. Excellent genetic stability was also observed in continuous passage culture. Therefore, strain KTU-P8DCA has enormous potential for use in bioremediation of sites heavily contaminated with DCA. In the future, our strategy for pathway construction and optimization is expected to be developed as a standard pipeline for creating a wide variety of new contaminants-mineralizing microorganisms. The present study also highlights the power of synthetic biology in creating novel degraders for environmental remediation.

Metformin pre-treatment of stem cells from human exfoliated deciduous teeth promotes migration and angiogenesis of human umbilical vein endothelial cells for tissue engineering.

BACKGROUND AIMS: Stem cells from human exfoliated deciduous teeth (SHED) play a significant role in tissue engineering and regenerative medicine. Angiogenesis is crucial in tissue regeneration and a primary target of regenerative medicine. As a first-line anti-diabetic drug, metformin demonstrates numerous valuable impacts on stem cells. This study aimed to explore metformin's impact and mechanism of action on SHED-mediated angiogenesis. METHODS: First, cell proliferation; flow cytometry; osteogenic, adipogenic and chondrogenic induction; and proteomics analyses were conducted to explore the role of metformin in SHED. Subsequently, migration and tube formation assays were used to evaluate chemotaxis and angiogenesis enhancement by SHED pre-treated with metformin under co-culture conditions in vitro, and relative messenger RNA expression levels were determined by quantitative reverse transcription polymerase chain reaction. Finally, nude mice were used for in vivo tube formation assay, and sections were analyzed through immunohistochemistry staining with anti-human CD31 antibody. RESULTS: Metformin significantly promoted SHED proliferation as well as osteogenic, adipogenic and chondrogenic differentiation. Proteomics showed that metformin significantly upregulated 124 differentially abundant proteins involved in intracellular processes, including various proteins involved in cell migration and angiogenesis, such as MAPK1. The co-culture system demonstrated that SHED pre-treated with metformin significantly improved the migration and angiogenesis of human umbilical vein endothelial cells. In addition, SHED pre-treated with metformin possessed greater ability to promote angiogenesis in vivo. CONCLUSIONS: In summary, the authors' findings illustrate metformin's mechanism of action on SHED and confirm that SHED pre-treated with metformin exhibits a strong capacity for promoting angiogenesis. This helps in promoting the application of dental pulp-derived stem cells pre-treated with metformin in regeneration engineering.