In this issue, we recommend Professor Chen Guoqiang's team, Director of the Center for Synthesis and Systems Biology at Tsinghua University, for publicationMetabolic EngineeringPrevious article: Engineering low-salt growth Halomonas Bluephagenesis for cost-effective bioproduction combined with adaptive evolution 。 This study used atmospheric pressure room temperature plasma technology (ARTP) to randomly mutate cyanobacteria and screen for strains that grow well at low salt concentrations. Fermentation of the obtained advantageous strains to produce PHA achieved a dual benefit of high yield and cost reduction. Comparing mutant strains with wild-type genotypes, salt stress-related genes of halophilic host strains were revealed.

Polyhydroxyalkanoates (PHA) are polymers used in bacteria for energy storage and can be developed into green biodegradable plastics. It possesses the physical and chemical properties of chemical plastics, but also has a series of properties such as biodegradability, biocompatibility, optical activity, and gas separation. The PHA producing strain, Halophilic Monomonas TD01, is a halophilic bacterium isolated from Lake Edingel in Xinjiang, China. It can grow rapidly under non sterile conditions at high pH and high salt concentration. Although high-performance strains of cyanobacteria have been modified in areas such as expression vectors and promoters, there are still many unknowns in their salt tolerance regulation. Reverse engineering of Blue Crystal Salt Cell Bacteria, such as low salt and high pH survival, can further reduce the cost of salt wastewater treatment, achieve higher cell separation and PHA purification.

However, due to the different allocation strategies of cellular proteins involved in biological process control in response to external pressures such as osmotic pressure of salinity, heat shock, UV irradiation, etc., rational design of metabolic networks to regulate salt stress poses certain difficulties. Atmospheric pressure room temperature plasma technology (ARTP) is a genome-wide random mutagenesis method. As the mechanism of microbial salt tolerance involves a system level regulatory network, ARTP that can achieve genome-wide random mutagenesis is more suitable for the chassis modification of cyanobacteria.

Researchers used wild-type Pseudomonas aeruginosa TD01 and three recombinant strains TDH4, TD68, and TD68-194 as starting strains to verify their growth status in high concentration (50g/L NaCl) and low concentration (10g/L NaCl) salt environments. The results showed that all strains did not form colonies in low salt concentration plates (Figure 2). ARTP mutagenesis was performed on these 4 strains of bacteria for 5 minutes, and the mortality curve showed that the optimal treatment time was 3 minutes (Figure 2). Four strains of bacteria that grow the fastest at low salt concentrations were selected from the library after mutagenesis, and a second round of mutagenesis was performed to obtain four strains: TD01A₂B₅, TDH4A₁B₅, TD68A₂B₃and TD68-194A₁B₅(Figure 2). Among them, TDH4A₁B₅Bacteria have excellent production performance, with a cell dry weight of up to 11g/L and a PHA yield of 60% by mass.

For mutant strainsTDH4A₁B₅Genetic modification was carried out by adding phaCAB operon to enhance metabolic flux. After 40 hours of unsterilized fed batch fermentation in a 7L system, the production of PHB and P34HB increased by 21% and 36% respectively compared to the wild type (Figure 3). Further modification resulted in a 50% increase in methionine secretion rate and a 77% increase in exocrine secretion rate, respectively. Multiple analyses have shown its potential for application as a low salt concentration chassis bacterium (Figure 4). Through gene comparison, the salt stress regulation mechanism of halophilic host strains was revealed, including 101 genes related to osmotic pressure. More importantly, by using recombinant cyanobacteria TDH4A1B5, the cost of PHA in the 7L fermentation system was reduced by one-third, significantly improving its economic competitiveness (Figure 5).

picture1 This article presents a technical roadmap

picture2 ARTPMutation data

picture3 rightH. bluephagenesisTDH4A₁B₅Transforming to achieve high yieldsPHA

picture4 rightH. bluephagenesisTDH4A₁B₅The transformationApplied to the production of various proteins

picture5 Cost analysis of low salt fermentation and high salt fermentation

Paper link:

https://doi.org/10.1016/j.ymben.2023.08.001