Seok Hoon Hong

Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation.

Site-specific incorporation of nonstandard amino acids (NSAAs) into proteins enables the creation of biopolymers, proteins, and enzymes with new chemical properties, new structures, and new functions. To achieve this, amber (TAG codon) suppression has been widely applied. However, the suppression efficiency is limited due to the competition with translation termination by release factor 1 (RF1), which leads to truncated products. Recently, we constructed a genomically recoded Escherichia coli strain lacking RF1 where 13 occurrences of the amber stop codon have been reassigned to the synonymous TAA codon (rEc.E13.ΔprfA). Here, we assessed and characterized cell-free protein synthesis (CFPS) in crude S30 cell lysates derived from this strain. We observed the synthesis of 190 ± 20 μg/mL of modified soluble superfolder green fluorescent protein (sfGFP) containing a single p-propargyloxy-l-phenylalanine (pPaF) or p-acetyl-l-phenylalanine. As compared to the parent rEc.E13 strain with RF1, this results in a modified sfGFP synthesis improvement of more than 250%. Beyond introducing a single NSAA, we further demonstrated benefits of CFPS from the RF1-deficient strains for incorporating pPaF at two- and five-sites per sfGFP protein. Finally, we compared our crude S30 extract system to the PURE translation system lacking RF1. We observed that our S30 extract based approach is more cost-effective and high yielding than the PURE translation system lacking RF1, ∼1000 times on a milligram protein produced/

Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids

basis. Looking forward, using RF1-deficient strains for extract-based CFPS will aid in the synthesis of proteins and biopolymers with site-specifically incorporated NSAAs.

Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation. We evolved chromosomal aminoacyl-tRNA synthetases (aaRSs) with up to 25-fold increased protein production for p-acetyl-L-phenylalanine and p-azido-L-phenylalanine (pAzF). We also evolved aaRSs with tunable specificities for 14 nsAAs, including an enzyme that efficiently charges pAzF while excluding 237 other nsAAs. These variants enabled production of elastin-like-polypeptides with 30 nsAA residues at high yields (∼50 mg/L) and high accuracy of incorporation (>95%). This approach to aaRS evolution should accelerate and expand our ability to produce functionalized proteins and sequence-defined polymers with diverse chemistries.

Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1.

Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (>1 g/L) and long-lasting (>10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems (OTSs) has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids

Site‐specific incorporation of non‐standard amino acids (NSAAs) into proteins opens the way to novel biological insights and applications in biotechnology. Here, we describe the development of a high yielding cell‐free protein synthesis (CFPS) platform for NSAA incorporation from crude extracts of genomically recoded Escherichia coli lacking release factor 1. We used genome engineering to construct synthetic organisms that, upon cell lysis, lead to improved extract performance. We targeted five potential negative effectors to be disabled: the nuclease genes rna, rnb, csdA, mazF, and endA. Using our most productive extract from strain MCJ.559 (csdA− endA−), we synthesized 550±40 μg mL−1 of modified superfolder green fluorescent protein containing p‐acetyl‐L‐phenylalanine. This yield was increased to ∼1300 μg mL−1 when using a semicontinuous method. Our work has implications for using whole genome editing for CFPS strain development, expanding the chemistry of biological systems, and cell‐free synthetic biology.

Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-l-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.Cell-free protein synthesis allows for producing proteins without the need of a host organism, thus sparing the researcher experimental hassle. Here, the authors developed a cell-free synthesis method that enables incorporating non-standard amino acids in the product.

Probiotic Escherichia coli inhibits biofilm formation of pathogenic E. coli via extracellular activity of DegP

Abstract Colicins are antimicrobial proteins produced by Escherichia coli, which, upon secretion from the host, kill non-host E. coli strains by forming pores in the inner membrane and degrading internal cellular components such as DNA and RNA. Due to their unique cell-killing activities, colicins are considered viable alternatives to conventional antibiotics. Recombinant production of colicins requires co-production of immunity proteins to protect host cells; otherwise, the colicins are lethal to the host. In this study, we used cell-free protein synthesis (CFPS) to produce active colicins without the need for protein purification and co-production of immunity proteins. Cell-free synthesized colicins were active in killing model E. coli cells with different modes of cytotoxicity. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Both colicins E1 and E2 decreased the level of persister cells (metabolically dormant cell populations that are insensitive to antibiotics) by up to six orders of magnitude compared to that of the rifampin pretreated persister cells. This study finds that colicins can eradicate non-growing cells including persisters, and that CFPS is a promising platform for rapid production and characterization of toxic proteins.

Cordycepin induces apoptosis of human ovarian cancer cells by inhibiting CCL5-mediated Akt/NF-κB signaling pathway

Many chronic infections involve bacterial biofilms, which are difficult to eliminate using conventional antibiotic treatments. Biofilm formation is a result of dynamic intra- or inter-species interactions. However, the nature of molecular interactions between bacteria in multi-species biofilms are not well understood compared to those in single-species biofilms. This study investigated the ability of probiotic Escherichia coli Nissle 1917 (EcN) to outcompete the biofilm formation of pathogens including enterohemorrhagic E. coli (EHEC), Pseudomonas aeruginosa, Staphylococcus aureus, and S. epidermidis. When dual-species biofilms were formed, EcN inhibited the EHEC biofilm population by 14-fold compared to EHEC single-species biofilms. This figure was 1,100-fold for S. aureus and 8,300-fold for S. epidermidis; however, EcN did not inhibit P. aeruginosa biofilms. In contrast, commensal E. coli did not exhibit any inhibitory effect toward other bacterial biofilms. We identified that EcN secretes DegP, a bifunctional (protease and chaperone) periplasmic protein, outside the cells and controls other biofilms. Although three E. coli strains tested in this study expressed degP, only the EcN strain secreted DegP outside the cells. The deletion of degP disabled the activity of EcN in inhibiting EHEC biofilms, and purified DegP directly repressed EHEC biofilm formation. Hence, probiotic E. coli outcompetes pathogenic biofilms via extracellular DegP activity during dual-species biofilm formation.

Medium chain unsaturated fatty acid ethyl esters inhibit persister formation of Escherichia coli via antitoxin HipB

The chemokine, CCL5, is a key mediator for the recruitment of immune cells into tumors and tissues. Akt/NF-κB signaling is significantly activated by CCL5. However, the role of NF-κB inactivation in apoptosis induced by negative regulation of CCL5 remains unclear. Here, we analyzed the effect of cordycepin on NF-κB activity in SKOV-3 cells and found that cordycepin-mediated inhibition of NF-κB signaling induced apoptosis in SKOV-3 cells via the serial activation of caspases. In addition, immune-blotting analysis showed that CCL5 is highly expressed in SKOV-3 cells. In addition to activating caspases, we show that, cordycepin prevents TNF-α-induced increase in CCL5, Akt, NF-κB, and c-FLIPL activation and that CCL5 siRNA could inhibit Akt/NF-κB signaling. Moreover, cordycepin negatively regulated the TNF-α-mediated IκB/NF-κB pathway and c-FLIPL activation to promote JNK phosphorylation, resulting in caspase-3 activation and apoptosis. Also, we show that c-FLIPL is rapidly lost in NF-κB activation-deficient. siRNA mediated c-FLIP inhibition increased JNK. SP600125, a selective JNK inhibitor, downregulated p-JNK expression in cordycepin-treated SKOV-3 cells, leading to suppression of cordycepin-induced apoptosis. Thus, these results indicate that cordycepin inhibits CCL5-mediated Akt/NF-κB signaling, which upregulates caspase-3 activation in SKOV-3 cells, supporting the potential of cordycepin as a therapeutic agent for ovarian cancer.

METHODS FOR IMPROVED IN VITRO PROTEIN SYNTHESIS WITH PROTEINS CONTAINING NON STANDARD AMINO ACIDS

Persisters represent a small bacterial population that is dormant and that survives under antibiotic treatment without experiencing genetic adaptation. Persisters are also considered one of the major reasons for recalcitrant chronic bacterial infections. Although several mechanisms of persister formation have been proposed, it is not clear how cells enter the dormant state in the presence of antibiotics or how persister cell formation can be effectively controlled. A fatty acid compound, cis-2-decenoic acid, was reported to decrease persister formation as well as revert the dormant cells to a metabolically active state. We reasoned that some fatty acid compounds may be effective in controlling bacterial persistence because they are known to benefit host immune systems. This study investigated persister cell formation by pathogens that were exposed to nine fatty acid compounds during antibiotic treatment. We found that three medium chain unsaturated fatty acid ethyl esters (ethyl trans-2-decenoate, ethyl trans-2-octenoate, and ethyl cis-4-decenoate) decreased the level of Escherichia coli persister formation up to 110-fold when cells were exposed to ciprofloxacin or ampicillin antibiotics. RNA sequencing analysis and gene deletion persister studies elucidated that these fatty acids inhibit bacterial persistence by regulating antitoxin HipB. A similar persister cell reduction was observed for pathogenic E. coli EDL933, Pseudomonas aeruginosa PAO1, and Serratia marcescens ICU2-4 strains. This study demonstrates that fatty acid ethyl esters can be used to disrupt bacterial dormancy to combat persistent infectious diseases.