Genome reduction enhances production of polyhydroxyalkanoate and alginate oligosaccharide in Pseudomonas mendocina
Xu Fan1, Yiting Zhang1, Fengjie Zhao1
1Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China.
Abstract:
Pseudomonas mendocina NK-01 previously isolated by our lab is able to accumulate medium-chain-length polyhydroxyalkanoate (mcl-PHA) intracellularly and secrete alginate oligosaccharide (AO) to the extracellular milieu. The present study aimed at investigating whether improved production of mcl-PHA and AO by P. mendocina can be accomplished by genome reduction. In this study, 14 large genomic fragments accounting for 7.7% of the genome of P. mendocina NK-01 were sequentially deleted to generate a series of genome-reduced strains by an upp-based markerless knockout method. As a result, the intracellular ATP/ADP ratio of the strain NKU421 with the largest deletion improved by 11 times compared to NK-01. More importantly, the mcl-PHA and AO yields of NKU421 increased by 114.8% and 27.8%, respectively. Enhancing mcl-PHA and AO production by NKU421 may be attributed to improved transcriptional levels of PHA synthase genes and AO secretion-related genes. The present study suggests that rational reduction of bacterial genome is a feasible approach to construct an optimal chassis for enhanced production of bacterial metabolites. In the future, further reduction of the NKU421 genome can be expected to create high-performance chassis for the development of microbial cell factories.
Insights
Genome reduction in Pseudomonas mendocina NK-01 enhanced production of medium-chain-length polyhydroxyalkanoate (mcl-PHA) and alginate oligosaccharide (AO). This strategy creates a more efficient microbial chassis for metabolite production.
Area of Science:
- Microbiology
- Synthetic Biology
- Metabolic Engineering
Background:
- Pseudomonas mendocina NK-01 naturally produces medium-chain-length polyhydroxyalkanoate (mcl-PHA) and alginate oligosaccharide (AO).
- Optimizing microbial strains for enhanced metabolite yields is crucial for industrial biotechnology.
Purpose of the Study:
- To investigate the efficacy of genome reduction in improving mcl-PHA and AO production in P. mendocina NK-01.
- To engineer a reduced-genome P. mendocina strain as an optimized chassis for metabolite overproduction.
Main Methods:
- Sequential deletion of 14 large genomic fragments (7.7% of genome) using an upp-based markerless knockout system.
- Generation of a series of genome-reduced strains, culminating in strain NKU421.
- Analysis of intracellular ATP/ADP ratios and metabolite yields in engineered strains.
Main Results:
- The genome-reduced strain NKU421 exhibited an 11-fold increase in intracellular ATP/ADP ratio compared to the wild-type NK-01.
- mcl-PHA yield increased by 114.8% and AO yield by 27.8% in strain NKU421.
- Enhanced production is potentially linked to upregulated PHA synthase and AO secretion genes.
Conclusions:
- Rational genome reduction is a viable strategy for constructing optimized microbial chassis.
- Genome-reduced P. mendocina NKU421 demonstrates significantly improved yields of mcl-PHA and AO.
- Further genome reduction holds potential for developing high-performance microbial cell factories.
