Overview
Lignin is the only polymer with aromatic skeleton, which has the potential to produce fuels, bio-based materials and aromatic compounds. This will not only help to alleviate the fossil fuel crisis and the climate crisis, but also develop a green bio-refining approach. In recent years, the biological utilization of lignin has attracted wide attention because of its advantages of low cost, environmental friendliness and mild reaction conditions. However, industrial catalyst strategy needs precious metal catalyst, which is expensive and harsh. However, the enzyme catalysis strategy based on microorganism has low degradation efficiency due to the limited enzyme expression of a single bacterium, which is difficult to meet the application requirements.
Chassis selection
NPU-China found that Pseudomonas putida can produce manganese oxide with lignin degradation activity by secreting lignin degradation enzyme and manganese oxidation, and catalyze lignin of macromolecules into aromatic compounds of small molecules, which is of great significance to environmental protection, green chemistry, energy, medicine and other fields. In addition, compared with other fungi, Pseudomonas putida has a short growth cycle and low large-scale culture conditions, so it is an ideal cell factory to construct efficient lignocellulose degradation. However, the ability of Pseudomonas putida to secrete extracellular enzymes and produce corresponding manganese oxidation is weak, so it is difficult to obtain high degradation efficiency.
Gene selection
In order to solve the problem, we overexpressed a series of genes related to manganese oxidation through expression plasmid, and at the same time, we reformed the promoter element of cytochrome c mature operon through genetic engineering technology, which greatly increased the expression of MnP, Lac and DyP, and increased the expression of strong oxidizing manganese oxide, so as to realize efficient biosynthesis of nano-enzyme and effective depolymerization and utilization of lignin.
The pathway of bacteria-mediated manganese oxidation is extremely complicated. Generally speaking, its mode of action can be divided into two types: (1) biological induction, which is beneficial to manganese oxidation by changing the surrounding microenvironment (such as pH) through bacterial metabolic activities, or inducing extracellular superoxide through bacterial-bacterial interaction, and then oxidizing Mn (Ⅱ); (2) Biological control, that is, the oxidation process of manganese is controlled by the enzyme system of microorganisms. At present, scientists generally believe that biological control plays a key role in the process of manganese oxidation of Pseudomonas putida, and multicopper oxidase (MCO), manganese peroxidases (MnP) and manganese catalase (MnC) may be the most widely involved natural enzymes. In addition, genes related to flagella synthesis, protein transport, two-component regulation system, cytochrome C synthesis and carbon metabolism also affect the manganese oxidation of Pseudomonas putida to some extent. Therefore, our team chose MCO, MnP and MnC as the target genes respectively, and overexpressed the above genes through the expression plasmid pBBR1-MCS5. In addition, by transforming the promoter element of cytochrome c mature operon and overexpressing a series of related genes, the manganese oxidation ability of Pseudomonas putida was improved synergistically.
Expression vector Construction
In order to achieve the above goal, we cloned the genes of three related enzymes into pBBR1-MCS5 plasmid and overexpressed them in Pseudomonas putida. Because the synthesis process of cytochrome c is closely related to the manganese oxidation of bacteria, we also replaced the original promoter of cytochrome c with a more sensitive promoter element by genetic engineering, so as to realize the over-expression of the following nine genes related to cytochrome c maturity. This promoter can simultaneously regulate the expression of different cytochrome c mature enzymes (ccmA, ccmB, ccmC, ccmD, ccmE, ccmF, ccmG, ccmH and ccmI). The transformation of this promoter is beneficial to improve the ability of Pseudomonas putida to produce manganese oxide and produce more manganese oxides with stronger oxidation degree. In addition, the plasmid used contains gentamicin immune sequence, so that we can determine whether the constructed plasmid is successfully transferred into the host cell. At the same time, gentamicin was added to all media to ensure that the surviving cells contained our construction plasmid.
