Biosynthesis of Polyhydroxyalkanoate by Recombinant Bacteria: A Review

Polyhydroxyalkanoates (PHAs), a biodegradable polymer, has many industrial and medical applications such as heart valves, scaffold, suture and drug delivery. Because of their structural diversity and close analogy to plastics, PHAs have gained major interest over the world. Natural isolates, recombinant bacteria, and plants have all been found to improve the quality, quantity, and economics of PHA production. Their biodegradability makes them an especially appealing synthetic plastic substitute. PHB biosynthetic genes phbA, phbB, and phbC are grouped and systematised into a single phbCAB operon. In terms of alignment and clustering of the relevant genes, the PHB pathway varies greatly across bacterial genera. In this regard, the enzymes appear to have a high degree of sequence preservation. The structural studies further improve the mechanism of action of these enzymes and helped in improving and choosing the good candidates for increased production of PHB.

Surface-Displayed Amuc_1100 From Akkermansia muciniphila on Lactococcus lactis ZHY1 Improves Hepatic Steatosis and Intestinal Health in High-Fat-Fed Zebrafish

Fatty liver and intestinal barrier damage were widespread in most farmed fish, which severely restrict the development of aquaculture. Therefore, there was an urgent need to develop green feed additives to maintain host liver and intestinal health. In this study, a probiotic pili-like protein, Amuc_1100 (AM protein), was anchored to the surface of Lactococcus lactis ZHY1, and the effects of the recombinant bacteria AM-ZHY1 on liver fat accumulation and intestinal health were evaluated. Zebrafish were fed a basal diet, high-fat diet, and high-fat diet with AM-ZHY1 (108 cfu/g) or control bacteria ZHY1 for 4 weeks. Treatment with AM-ZHY1 significantly reduced hepatic steatosis in zebrafish. Quantitative PCR (qPCR) detection showed that the expression of the lipogenesis [peroxisome-proliferator-activated receptors (PPARγ), sterol regulatory element-binding proteins-1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase 1 (ACC1)] and lipid transport genes (CD36 and FABP6) in the liver were significantly downregulated (p < 0.05), indicating that AM-ZHY1 could reduce liver fat accumulation by inhibiting lipid synthesis and absorption. Moreover, supplementing AM-ZHY1 to a high-fat diet could significantly reduce serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, indicating that liver injury caused by high-fat diets was improved. The expression of tumor necrosis factor (TNF)-a and interleukin (IL)-6 in the liver decreased significantly (p < 0.05), while IL-1β and IL-10 did not change significantly in the AM-ZHY1 group. Compared to the high-fat diet-fed group, the AM-ZHY1 group, but not the ZHY1 group, significantly increased the expression of intestinal tight junction (TJ) proteins (TJP1a, claudina, claudin7, claudin7b, claudin11a, claudin12, and claudin15a; p < 0.05). Compared to the high-fat diet group, the Proteobacteria and Fusobacteria were significantly reduced and increased in the AM-ZHY1 group, respectively. In conclusion, the recombinant bacteria AM-ZHY1 has the capacity to maintain intestinal health by protecting intestinal integrity and improving intestinal flora structure and improving fatty liver disease by inhibiting lipid synthesis and absorption. This study will lay a foundation for the application of AM protein in improving abnormal fat deposition and restoring the intestinal barrier in fish.

SP-1, a Serine Protease from the Gut Microbiota, Influences Colitis and Drives Intestinal Dysbiosis in Mice

Increased protease activity has been linked to the pathogenesis of IBD. While most studies have been focusing on host proteases in gut inflammation, it remains unclear how to address the potential contribution of their bacterial counterparts. In the present study, we report a functional characterization of a newly identified serine protease, SP-1, from the human gut microbiota. The serine protease repertoire of gut Clostridium was first explored, and the specificity of SP-1 was analyzed using a combinatorial chemistry method. Combining in vitro analyses and a mouse model of colitis, we show that oral administration of recombinant bacteria secreting SP-1 (i) compromises the epithelial barrier, (ii) alters the microbial community, and (ii) exacerbates colitis. These findings suggest that gut microbial protease activity may constitute a valuable contributor to IBD and could, therefore, represent a promising target for the treatment of the disease.

Study on the Degradation Effect of Three Organophosphorus Hydrolase Mutant on Sarin

Organophosphorus hydrolase can effectively degrade organic phosphorus compounds such as sarin. In this study, we constructed a recombinant Bacillus subtilis mutant expressing organophosphorus hydrolase, measured the effect of the mutant on the degradation rate of nerve agent sarin, and selected the optimal mutation scheme. Three different hydrolase mutant genes, 257L, 257Y and 303T, were ligated to PMA0911 vector and transferred into Bacillus subtilis WB800 to construct the target recombinant strain successfully. The recombinant bacteria secreted the target protein by fermentation. The effect of enzyme protein on the degradation of sarin was determined by the benzidine method. The optimal mutant was screened, and its enzymatic performance was explored. The effects of three organophosphorus hydrolase mutants on the hydrolysis rate of sarin were detected. The results showed that the 257Y mutant accelerated the hydrolysis of sarin significantly. Point mutation can improve the enzyme activity of wild-type organophosphorus hydrolase to a certain extent, laying the foundation for subsequent in-depth research.

Male-biased Adult Production of the Striped Fruit Fly, Zeugodacus scutellata, by Feeding dsRNA Specific to Transformer-2

Sterile insect release technique (SIT) is effective for eradicating quarantine insects including various tephritid fruit flies. When SIT is used for fruit flies, it is challenging to remove females from sterile males due to oviposition-associated piercing damage. This study developed a sex transition technique by feeding double-stranded RNA (dsRNA) specific to a sex-determining gene, Transformer-2 (Zs-Tra2) of the striped fruit fly, Zeugodacus scutellata. Zs-Tra2 is homologous to other fruit fly orthologs. It is highly expressed in female adults. RNA interference (RNAi) of Zs-Tra2 by injecting or feeding its specific dsRNA to larvae significantly increased male ratio. Recombinant Escherichia coli cells expressing dsRNA specific to Zs-Tra2 were prepared and used to feed larvae to suppress Zs-Tra2 gene expression levels. When these recombinant bacteria were fed to larvae during the entire feeding stage, the test population was significantly male-biased. Some females treated with such recombinant E. coli exhibited mosaic morphological characters such as the presence of male-specific abdominal setae in females. This study proposes a novel technique by feeding dsRNA specific to Transformer-2 to reduce female production during mass-rearing of tephritid males for SIT.

Statistical noise from recombinant plasmids can be abated via complementation of a ribosomal protein gene deletion

The phenotypes conferred by recombinant plasmids upon host cells often exhibit variability between replicate populations. This statistical noise is mostly a consequence of adaptive evolution in response to fitness burdens imposed by the plasmids themselves. We developed a novel strategy, ‘ribosome pegging’, to exclude common unwanted mutations that benefit host cells at the expense of heterologous gene expression. Plasmids that constitutively co-expressed the fluorescent reporter tagRFP and ribosomal protein L23 (rplW) were used to transform Escherichia coli cells that lacked the essential chromosomal rplW gene. Cells within the population that expressed too little L23, or too much, were evidently inviable. Ribosome pegging obviates the need for antibiotics, thus facilitating the deployment of recombinant bacteria in uncontrolled environments. We show that ribosome-pegged E. coli carrying a plasmid that constitutively expresses L23 and an artificially evolved enzyme protects fruit flies from otherwise toxic doses of the insecticide malathion.