Po Ting Chen

Replicon-free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli.

Genetic manipulation of cells for desired traits is the most appreciable strategy implemented in the field of bioengineering. However, this approach closely relies on the use of plasmids and is commonly afflicted by the potential problem of plasmid instability and safety caution. Meanwhile, it may also lead to the spread of antibiotic‐resistant markers with replicons of plasmids to the environment. However, this issue has long been neglected. In this study, we have addressed these subjects by developing replicon‐free and markerless methods for chromosomal insertion of genes and controlled expression of genomic genes in Escherichia coli. For the former application, the integration vectors of conditional replication were incorporated with the prophage attachment site and duplicated FRT sites. Their utility was illustrated by site‐specific insertion of target genes, the endogenous dxs gene and three heterologous genes consisting of gps, crtI, and crtB, fused to T7 promoter into E. coli genome. For the latter application, the template vectors for promoter replacement were constructed to carry a DNA cassette containing the T7 promoter linked to a selective marker flanked with the FRT site. Subsequently, it was illustrated by replacement of the native promoter of chromosomal pckA by the T7 promoter. Finally, with the aid of FLP recombinase supplied from a helper plasmid, the regions containing replicon and/or selective markers in inserted DNAs were eliminated from integrants for both approaches. As a consequence, the expression of these five genes was subject to control by one response regulator, T7 RNA polymerase, in a regulon way, resulting in a high and stable production of lycopene in the cell. This result indicates the promise of developed methods for genome engineering in E. coli. Biotechnol. Bioeng.

One-Step Production of d-p-Hydroxyphenylglycine by Recombinant Escherichia coli Strains

The gene encoding d‐hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction. A positive clone was scored, and its nucleotide sequence was further analyzed. The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site. By highly expressing both d‐hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert dl‐hydroxyphenyl hydantoin (dl‐HPH) to d‐p‐hydroxyphenylglycine (d‐HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A. radiobacter NRRL B11291. Immobilizing the recombinant cells with κ‐carrageenan could also achieve a conversion of 93%, while A. radiobacter NRRL B11291 attained 20% within the same period of reaction time. These results illustrate the feasibility in employing recombinant E. coli to accomplish one‐step conversion of dl‐HPH to d‐HPG. In the process of improving d‐HPG production, d‐hydantoinase activity was increased 2.57‐fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate. It suggests that carbamoylase is the step setting the pace of the reaction. Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system. This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice. In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.

Strategy To Approach Stable Production of Recombinant Nattokinase in Bacillus subtilis

Bacillus subtilis (B. subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins. In this study, a shuttle vector was constructed by fusion of Staphylococcus aureus (S. aureus) plasmid pUB110 with Escherichia coli (E. coli) plasmid pUC18 and used for the expression of nattokinase in B. subtilis. The pUB110/pUC‐based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions. An initial attempt was made to eliminate the homologous site in the plasmid, whereas the stability of the resulting plasmid was not improved. In an alternative way, the pUC18‐derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E. coli. As a consequence, the pUB110/R6K‐based plasmid displayed full structural stability, leading to a high‐level production of recombinant nattokinase in the culture broth. This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid. Moreover, 2‐fold higher nattokinase production was obtained by B. subtilis strain carrying the pUB110/R6K‐based plasmid as compared to the cell with the pAMβ1‐derived vector, a plasmid known to have high structural stability. Overall, it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B. subtilis.

Medium Optimization for the Production of Recombinant Nattokinase by Bacillus subtilis Using Response Surface Methodology

Nattokinase is a potent fibrinolytic enzyme with the potential for fighting cardiovascular diseases. Most recently, a new Bacillus subtilis/Escherichia coli (B. subtilis/E. coli) shuttle vector has been developed to achieve stable production of recombinant nattokinase in B. subtilis (Chen; et al. 2007 , 23, 808–813). With this developed B. subtilis strain, the design of an optimum but cost‐effective medium for high‐level production of recombinant nattokinase was attempted by using response surface methodology. On the basis of the Plackett‐Burman design, three critical medium components were selected. Subsequently, the optimum combination of selected factors was investigated by the Box‐Behnken design. As a result, it gave the predicted maximum production of recombinant nattokinase with 71 500 CU/mL for shake‐flask cultures when the concentrations of soybean hydrolysate, potassium phosphate, and calcium chloride in medium were at 6.100, 0.415, and 0.015%, respectively. This was further verified by a duplicated experiment. Moreover, the production scheme based on the optimum medium was scaled up in a fermenter. The batch fermentation of 3 L was carried out by controlling the condition at 37 °C and dissolved oxygen reaching 20% of air saturation level while the fermentation pH was initially set at 8.5. Without the need for controlling the broth pH, recombinant nattokinase production with a yield of 77 400 CU/mL (corresponding to 560 mg/L) could be obtained in the culture broth within 24 h. In particular, the recombinant B. subtilis strain was found fully stable at the end of fermentation when grown on the optimum medium. Overall, it indicates the success of this experimental design approach in formulating a simple and cost‐effective medium, which provides the developed strain with sufficient nutrient supplements for stable and high‐level production of recombinant nattokinase in a fermenter.

Strategy for stable and high-level expression of recombinant trehalose synthase in Escherichia coli.

Trehalose is a nonreducing disaccharide and has a wide range of applications in food and biorelated industry. This sugar can be synthesized from maltose in one step by trehalose synthase. In this study, we attempted to overproduce trehalose synthase from Picrophilus torridus (PTTS), a thermoacidophilic archaea, in Escherichia coli . However, overproduction of PTTS was hampered when the T7 promoter-driven PTTS gene (PT7-PTTS) on a multicopy plasmid was employed in E. coli . The factors limiting PTTS production were identified in a systematic way, including the codon bias, plasmid instability, a redundant gene copy, a high basal level of PTTS, and metabolic burden resulting from the mutlicopy plasmid DNA and antibiotics. To overcome these difficulties, an E. coli strain was developed with insertion of PT7-PTTS into the chromosome and enhanced expression of genomic argU tRNA and ileX tRNA genes. Without the selective pressure, the constructed producer strain was able to produce a stable and high-level production of recombinant PTTS. Overall, we proposed a simple and effective method to address the issue that is most commonly raised in overproduction of heterologous proteins by E. coli .

Marker-free chromosomal expression of foreign and native genes in Escherichia coli.

Genetic manipulation of Escherichia coli strains for desired traits is the most applied strain engineering approach in industrial applications. For chromosomal insertion of genes and controlled expression of genomic genes in E. coli, the replicon-free and markerless method is described based on a series of conditional-replication plasmids called phage-integration vectors. They mainly carry the multiple cloning site and the prophage attachment site, which are sandwiched by two FRT sites. With the aid of the phage integrase from conditional-replication helper plasmids, the passenger genes of either foreign or native type incorporated into the integration vectors can be specifically integrated into bacterial genome at the prophage attachment site. Finally, the inserted DNA containing replicon and/or selective markers in integrants can be eliminated by the act of the FLP recombinase provided from a conditional-replication helper plasmid.

Biorefining of protein waste for production of sustainable fuels and chemicals

To mitigate the climate change caused by CO2 emission, the global incentive to the low-carbon alternatives as replacement of fossil fuel-derived products continuously expands the need for renewable feedstock. There will be accompanied by the generation of enormous protein waste as a result. The economical viability of the biorefinery platform can be realized once the surplus protein waste is recycled in a circular economy scenario. In this context, the present review focuses on the current development of biotechnology with the emphasis on biotransformation and metabolic engineering to refine protein-derived amino acids for production of fuels and chemicals. Its scope starts with the explosion of potential feedstock sources rich in protein waste. The availability of techniques is applied for purification and hydrolysis of various feedstock proteins to amino acids. Useful lessons are leaned from the microbial catabolism of amino acids and lay a foundation for the development of the protein-based biotechnology. At last, the future perspective of the biorefinery scheme based on protein waste is discussed associated with remarks on possible solutions to overcome the technical bottlenecks.