Systematic metabolic engineering of Escherichia coli for the enhanced production of cinnamaldehyde.

Cinnamaldehyde (CAD) derived from cinnamon bark has received much attention for its potential as a nematicide and food additive. Previously, we have succeeded in developing an Escherichia coli strain (YHP05) capable of synthesizing cinnamaldehyde; however, the production titer (75 mg/L) was not sufficient for commercialization. Herein, to develop an economical and sustainable production bioprocess, we further engineered the YHP05 strain for non-auxotrophic, antibiotic-free, inducer-free hyperproduction of CAD using systematic metabolic engineering. First, the conversion of trans-cinnamic acid (t-CA) to CAD was improved by the co-expression of carboxylic acid reductase and phosphopantetheinyl transferase (PPTase) genes. Second, to prevent the spontaneous conversion of CAD to cinnamyl alcohol, 10 endogenous reductase and dehydrogenase genes were deleted. Third, all expression cassettes were integrated into the chromosomal DNA using an auto-inducible system for antibiotic- and inducer-free production. Subsequently, to facilitate CAD production, available pools of cofactors (NADPH, CoA, and ATP) were increased, and acetate pathways were deleted. With the final antibiotic-, plasmid-, and inducer-free strain (H-11MPmR), fed-batch cultivations combined with in situ product recovery (ISPR) were performed, and the production titer of CAD as high as 3.8 g/L could be achieved with 49.1 mg/L/h productivity, which is the highest CAD titer ever reported.

Production of Cinnamaldehyde through Whole-Cell Bioconversion from trans-Cinnamic Acid Using Engineered Corynebacterium glutamicum.

Cinnamaldehyde (CAD) has various applications in foods and pharmaceuticals and has gained prominence as a potent nematicide in agricultural research owing to its nematicidal activity. However, conventional methods of CAD production, including extraction from plants or organic chemical synthesis, are environmentally hazardous and limit its utilization for downstream applications. Here, we engineered Corynebacterium glutamicum as a whole-cell biocatalyst for the efficient bioconversion of trans-cinnamic acid (t-CA) into CAD. An expression module of Mycobacterium phlei carboxylic acid reductase was constructed for the conversion of t-CA to CAD. Additionally, the putative dehydrogenase-related genes (dkgA, adhC, and cg1176) responsible for the conversion of CAD to cinnamyl alcohol were deleted from the engineered C. glutamicum strain to prevent the loss of CAD. Furthermore, as the conversion is NADPH-dependent, we investigated the conversion efficiency by exchanging the putative promoter region for the zwf gene, which encodes glucose-6-phosphate dehydrogenase, with a strong promoter to increase the NADPH pool. Finally, a bioconversion platform using C. glutamicum as a whole-cell biocatalyst was developed by deleting the vdh gene, which is involved in the reverse conversion of CAD to t-CA. Taken together, a 100% conversion yield of 1.1 g/L CAD from 1.2 g/L t-CA was obtained within 30 min.

Enhanced production of styrene by engineered Escherichia coli and in situ product recovery (ISPR) with an organic solvent.

BACKGROUND: Styrene is a large-volume commodity petrochemical, which has been used in a wide range of polymer industry as the main building block for the construction of various functional polymers. Despite many efforts to produce styrene in microbial hosts, the production titers are still low and are not enough to meet the commercial production of styrene. RESULTS: Previously, we developed a high L-phenylalanine producer (E. coli YHP05), and it was used as a main host for de novo synthesis of styrene. First, we introduced the co-expression system of phenylalanine-ammonia lyase (PAL) and ferulic acid decarboxylase (FDC) genes for the synthesis of styrene from L-phenylalanine. Then, to minimize cell toxicity and enhance the recovery of styrene, in situ product recovery (ISPR) with n-dodecane was employed, and culture medium with supplementation of complex sources was also optimized. As a result, 1.7 ± 0.1 g/L of styrene was produced in the flask cultures. Finally, fed-batch cultivations were performed in lab-scale bioreactor, and to minimize the loss of volatile styrene during the cultivation, three consecutive bottles containing n-dodecane were connected to the air outlet of bioreactor for gas-stripping. To conclude, the total titer of styrene was as high as 5.3 ± 0.2 g/L, which could be obtained at 60 h. CONCLUSION: We successfully engineered E. coli strain for the de novo production of styrene in both flask and fed-batch cultivation, and could achieve the highest titer for styrene in bacterial hosts reported till date. We believe that our efforts in strain engineering and ISPR strategy with organic solvent will provide a new insight for economic and industrial production of styrene in a biological platform.

Conformation-switchable helical polypeptide eliciting selective pro-apoptotic activity for cancer therapy.

Artificial cationic helical peptides possess an enhanced cell-penetrating property. However, their cell-penetrability is not converted by cellular environmental changes resulting in nonspecific uptake. In this study, pH-sensitive anion-donating groups were added to a helical polypeptide to simultaneously achieve tumor targeting and pro-apoptotic activity. The mitochondria-destabilizing helical polypeptide undergoing pH-dependent conformational transitions selectively targeted cancer cells consequently disrupting mitochondrial membranes and subsequently inducing apoptosis. This work presents a promising peptide therapeutic system for cancer therapy.

Metabolic engineering of Escherichia coli for the production of cinnamaldehyde.

BACKGROUND: Plant parasitic nematodes are harmful to agricultural crops and plants, and may cause severe yield losses. Cinnamaldehyde, a volatile, yellow liquid commonly used as a flavoring or food additive, is increasingly becoming a popular natural nematicide because of its high nematicidal activity and, there is a high demand for the development of a biological platform to produce cinnamaldehyde. RESULTS: We engineered Escherichia coli as an eco-friendly biological platform for the production of cinnamaldehyde. In E. coli, cinnamaldehyde can be synthesized from intracellular L-phenylalanine, which requires the activities of three enzymes: phenylalanine-ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), and cinnamoyl-CoA reductase (CCR). For the efficient production of cinnamaldehyde in E. coli, we first examined the activities of enzymes from different sources and a gene expression system for the selected enzymes was constructed. Next, the metabolic pathway for L-phenylalanine biosynthesis was engineered to increase the intracellular pool of L-phenylalanine, which is a main precursor of cinnamaldehyde. Finally, we tried to produce cinnamaldehyde with the engineered E. coli. According to this result, cinnamaldehyde production as high as 75 mg/L could be achieved, which was about 35-fold higher compared with that in the parental E. coli W3110 harboring a plasmid for cinnamaldehyde biosynthesis. We also confirmed that cinnamaldehyde produced by our engineered E. coli had a nematicidal activity similar to the activity of commercial cinnamaldehyde by nematicidal assays against Bursaphelenchus xylophilus. CONCLUSION: As a potential natural pesticide, cinnamaldehyde was successfully produced in E. coli by construction of the biosynthesis pathway and, its production titer was also significantly increased by engineering the metabolic pathway of L-phenylalanine.

High-Level Production of Human Papillomavirus (HPV) Type 16 L1 in Escherichia coli.

Human papillomavirus (HPV), a non-enveloped, double-stranded DNA tumor virus, is a primary etiological agent of cervical cancer development. As a potential tool for prophylactic vaccination, the development of virus-like particles (VLPs) containing the HPV16 L1 capsid protein is highly desired. In this study, we developed a high-level expression system of the HPV16 L1 in Escherichia coli for the purpose of VLP development. The native gene of HPV16 L1 has many rare codons that cause the early termination of translation and result in the production of truncated forms. First, we optimized the codon of the HPV16 L1 gene to the preferable codons of E. coli, and we succeeded in producing the full-size HPV16 L1 protein without early termination. Next, to find the best host for the production of HPV16 L1, we examined a total of eight E. coli strains, and E. coli BL21(DE3) with the highest yield among the strains was selected. With the selected host-vector system, we did a fed-batch cultivation in a lab-scale bioreactor. Two different feeding solutions (complex and defined feeding solutions) were examined and, when the complex feeding solution was used, a 6-fold higher production yield (4.6 g/l) was obtained compared with that with the defined feeding solution.

Rapid isolation of antibody from a synthetic human antibody library by repeated fluorescence-activated cell sorting (FACS).

Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS). First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv) was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show K(D) values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼ 10(6)). These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required.