Yun-Peng Chao

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.

Potential production platform of n-butanol in Escherichia coli.

We proposed a potential production platform of n-butanol in Escherichia coli. First, a butyrate-conversion strain was developed by removal of undesired genes and recruiting endogenous atoDA and Clostridium adhE2. Consequently, this E. coli strain grown on the M9 mineral salt with yeast extract (M9Y) was shown to produce 6.2g/L n-butanol from supplemented butyrate at 36h. The molar conversion yield of n-butanol on butyrate reaches 92%. Moreover, the production platform was advanced by additional inclusion of a butyrate-producing strain. This strain was equipped with a pathway comprising atoDA and heterologous genes for the synthesis of butyrate. Without butyrate, the butyrate-conversion and the butyrate-producing strains were co-cultured in M9Y medium and produced 5.5g/L n-butanol from glucose at 24h. The production yield on glucose accounts for 69% of the theoretical yield. Overall, it indicates a promise of the developed platform for n-butanol production in E. coli.

Stringent regulation and high-level expression of heterologous genes in Escherichia coli using T7 system controllable by the araBAD promoter.

The recombinant Eschreichia coli strain BL21 (BAD) was constructed to carry a chromosomal copy of T7 gene 1 fused to the araBAD promoter. To further characterize this expression system, strain BL21 (BAD) was transformed with the plasmid containing the carbamoylase gene from Agrobacterium radiobacter driven by the T7 promoter. Upon induction with l‐arabinose, recombinant cells produced 100‐fold increase in carbamoylase activity in comparison with uninduced cells on M9 semidefined medium plus glycerol. This protein yield accounts for 30% of total cell protein content. In addition, it was found that after 100 generations the plasmid harboring the carbamoylase gene remained firmly stable in strain BL21 (BAD), but its stability dropped to only 20–30% in strain BL21 (DE3), a commercial strain bearing T7 gene 1 regulated by the lacUV5 promoter in its chromosome. In an attempt to enhance the total protein yield, fed‐batch fermentation process was carried out using a two‐stage feeding strategy to compartmentalize cell growth and protein synthesis. In the batch fermentation stage, the culture was grown on glucose to reach the stationary growth phase. Subsequently, glycerol was fed to the culture broth and l‐arabinose was augmented to induce protein production when cells entered the late log growth phase. As a result, a carbamoylase yield corresponding to 5525 units was obtained, which amounts to a 337‐fold increase over that achieved on a shake‐flask scale. Taken together, these results illustrate the practical usefulness of T7 system under control of the araBAD promoter for heterologous protein production.

Enhanced conversion rate of L-phenylalanine by coupling reactions of aminotransferases and phosphoenolpyruvate carboxykinase in Escherichia coli K-12.

In Escherichia coli, aspartate aminotransferase (encoded by aspC) and aromatic amino acid aminotransferase (encoded by tyrB) share overlapping substrate specificity in the syntheses of aromatic amino acids. Through the transamination reactions catalyzed by AspC or TyrB, l‐phenylalanine (l‐Phe) can be produced from phenylpyruvate with aspartic acid as the amino donor. To modulate and enhance the production levels of proteins, both aspC and tyrB were subcloned into a runaway‐replication vector. As a result, the specific activities of AspC and TyrB obtained showed 65‐fold and 50‐fold increases, respectively, compared with the wild‐type level. Employing resting cells of AspC‐ and TyrB‐overproducing E. coli K‐12 strains for l‐Phe productions resulted in molar conversion yields of 70% and 55%, respectively. With an additional introduction of phosphoenolpyruvate carboxykinase (encoded by pck) into the transamination reactions, the conversion yields were improved to 93% from 70% and to 75% from 55% in a relatively short time. These results account for more than an 8‐fold increase in productivity, as compared to the previous report (Calton et al., 1985 ). In addition, a four‐run reuse of the recombinant cells for l‐Phe production gave a total yield of 91 g/L with a 93% conversion.

Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli ☆

With L-aspartate (L-Asp) as the amino donor, L-phenylalanine (L-Phe) can be prepared from phenylpyruvate (PPA) via an amination reaction mediated by aminotransferase (encoded by aspC). On the other hand, L-Asp can be produced by an aspartase (encoded by aspA) -catalyzed reaction using fumaric acid as substrate. To overproduce aspartase in Escherichia coli, the aspA gene was cloned and overexpressed 180 times over the wild-type level. The use of AspA-overproducing E. coli strain for L-Asp production exhibited an 83% conversion, approaching to the theoretical yield, whereas the wild-type strain obtained scarcely L-Asp. Furthermore, the recombinant strain overproducing both AspA and AspC was able to produce L-Asp and L-Phe simultaneously by using fumaric acid and PPA as substrates. As a result, the conversion yields obtained for L-Asp and L-Phe were 78% and 85%, respectively. In sharp contrast, the wild-type strain attained a conversion of L-Phe less than 15% and an undetectable level of L-Asp. This result illustrates a potential and attractive process to yield both L-Asp and L-Phe by coupling AspA and AspC. A further study on the repeated use of the recombinant strain immobilized with calcium alginate showed that after eight batch runs L-Asp conversion maintained roughly constant (around 75%), whereas L-Phe conversion dropped to 65% from 81%. This result indicates the stability of AspA being superior to AspC.

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.

Systematic Approach To Engineer Escherichia coli Pathways for Co-utilization of a Glucose–Xylose Mixture

Glucose and xylose are two major sugars of lignocellulosic hydrolysate. The regulatory program of catabolite repression in Escherichia coli dictates the preferred utilization of glucose over xylose, which handicaps the development of the lignocellulose-based fermentation process. To co-utilize a glucose-xylose mixture, the E. coli strain was manipulated by pathway engineering in a systematic way. The approach included (1) blocking catabolite repression, (2) enhancing glucose transport, (3) increasing the activity of the pentose phosphate pathway, and (4) eliminating undesirable pathways. Moreover, the ethanol synthetic pathway from Zymomonas mobilis was introduced into the engineered strain. As a consequence, the resulting strain was able to simultaneously metabolize glucose and xylose and consume all sugars (30 g/L each) in 16 h, leading to 97% of the theoretical ethanol yield. Overall, this indicates that this approach is effective and straightforward to engineer E. coli for the desired trait.

Overproduction of D-hydantoinase and carbamoylase in a soluble form in Escherichia coli

Abstract The production of d-hydantoinase and carbamoylase from Agrobacterium radiobacter NRRL B11291 using T7 and trc promoters, respectively, was found to cause protein aggregates in Escherichia coli. We initiated a systematic study aimed at overproducting these two proteins in a soluble form. As a result, the protein aggregate from carbamoylase overproduction could be alleviated with the aid of GroEL/GroES. In contrast, the production of a high level of d-hydantoinase in an active form can be achieved at low temperature (25 °C) or by the coproduction of DnaJ/DnaK. Overall, with such approaches both recombinant proteins gain more than a four-fold increase in enzyme activity. In addition, by fusion with thioredoxin, d-hydantoinase activity can be increased 25% more than the unfused counterpart in the presence of DnaJ/DnaK. These results indicate the success of our approaches to overproducing d-hydantoinase and carbamoylase in a soluble form in E. coli.

Effect of Pleurotus eryngii stalk residue on the oxidative status and meat quality of broiler chickens.

Pleurotus eryngii stalk residue (PESR) is a byproduct of the edible portion of the fruiting body. The present study was conducted to evaluate the effects of PESR on the oxidative status and meat quality of broilers. Two hundred fifty 1-d-old male broilers (Arbor Acre) were evenly divided by gender and randomly allocated into control (corn-soybean meal diet) or 1.0, 5.0, 10.0, or 20.0 g/kg dried PESR groups. The results revealed that at 35 d, the dried PESR groups displayed a significantly increased water-holding capacity and decreased storage loss of breast and thigh fillets when compared to the control group. Regarding fillets color, the L* (lightness) values were lower and the a* (redness) and b* (yellowness) values were higher following dried PESR supplementation. In 5.0-20.0 g/kg PESR supplementation groups, the activities of antioxidative enzymes were significantly elevated in serum, liver, spleen, and fillet tissues when compared to control group. Additionally, malondialdehyde production was slightly decreased in the PESR supplementation groups. Lower crude fat contents were observed in fillet tissues of 5.0-20.0 g/kg PESR groups when compared with the control group. In conclusion, PESR may potentially be used as an antioxidant to decrease lipid peroxidation and improve meat quality in broilers.

Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9.

Metabolic engineering often necessitates chromosomal integration of multiple genes but integration of large genes into Escherichia coli remains difficult. CRISPR/Cas9 is an RNA‐guided system which enables site‐specific induction of double strand break (DSB) and programmable genome editing. Here, we hypothesized that CRISPR/Cas9‐triggered DSB could enhance homologous recombination and augment integration of large DNA into E. coli chromosome. We demonstrated that CRISPR/Cas9 system was able to trigger DSB in >98% of cells, leading to subsequent cell death, and identified that mutagenic SOS response played roles in the cell survival. By optimizing experimental conditions and combining the λ‐Red proteins and linear dsDNA, CRISPR/Cas9‐induced DSB enabled homologous recombination of the donor DNA and replacement of lacZ gene in the MG1655 strain at efficiencies up to 99%, and allowed high fidelity, scarless integration of 2.4, 3.9, 5.4, and 7.0 kb DNA at efficiencies approaching 91%, 92%, 71%, and 61%, respectively. The CRISPR/Cas9‐assisted gene integration also functioned in different E. coli strains including BL21 (DE3) and W albeit at different efficiencies. Taken together, our methodology facilitated precise integration of dsDNA as large as 7 kb into E. coli with efficiencies exceeding 60%, thus significantly ameliorating the editing efficiency and overcoming the size limit of integration using the commonly adopted recombineering approach. Biotechnol. Bioeng. 2017;114: 172–183.