Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway

Abstract Numerous high‐value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec‐based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super‐secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat‐dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L−1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.

problems (Balasundaram, Harrison, & Bracewell, 2009). In addition, most biopharmaceuticals contain disulfide bonds, and these can only form in the periplasm in Gram-negative bacteria.
Efficient E. coli secretion-based systems form the basis for a range of industrial biopharmaceutical production platforms; however, many target proteins fail to be exported to the periplasm because the standard export method (via the Sec pathway) is only capable of transporting proteins in an unfolded state (Natale, Bruser, & Driessen, 2008). Many heterologous proteins pose problems due to rapid folding in the cytoplasm. However, most bacteria possess a second protein export pathway, known as the Tat pathway that has unique capabilities and two major advantages over the standard Sec pathway. First, the Tat pathway exports fully folded proteins, and has already been shown to export a number of "Sec-incompatible" heterologous molecules (DeLisa, Tullman, & Georgiou, 2003;Thomas, Daniel, Errington, & Robinson, 2001). Moreover, the Tat system can transport substrates up to 150 kDa in size and it even exports some natural substrates in a preassembled dimeric form; Tat therefore has clear potential for export of relatively complex molecules (Rodrigue, Chanal, Beck, Muller, & Wu, 1999). Secondly, the Tat pathway has an inbuilt "quality control" system, whereby it preferentially exports correctly folded proteins-it has been shown to secrete a range of heterologous proteins, including several biopharmaceuticals (Alanen et al., 2015;Matos et al., 2014), yet quantitatively reject virtually every misfolded protein tested to date (DeLisa et al., 2003;Matos, Robinson, & Di Cola, 2008;Richter & Bruser, 2005;Robinson et al., 2011). It thus has potential for the export of correctly folded, highly active target proteins with minimal heterogeneity; that is, it simultaneously provides both a means of exporting the product to the periplasm and increasing product "quality," thereby decreasing DSP costs.
To date, the Tat system has not been used for industrial secretionbased strategies, primarily because product yields have been low. This is partly due to the low abundance of the Tat apparatus when compared to the Sec system, which in turn reflects the fact that relatively few proteins are naturally transported by the Tat system (Tullman-Ercek et al., 2007). It has been shown that over-expression of the TatABC proteins from a second plasmid can boost export of a heterologous target protein , but the use of dual plasmids has clear disadvantages for industrial applications (Barrett, Ray, Thomas, Robinson, & Bolhuis, 2003).
Here, we show that over-expression of the tatABC genes from the chromosome leads to a major enhancement of Tat export capacity, yielding strains that have clear potential for industrial applications.  Barrett et al. (2003) ΔtatABCDE MC4100 strain lacking tatABCED genes, Ara R Barrett et al. (2003) pDOC-K Gene doctoring plasmid carrying ampicillin and kanamycin resistance markers Lee et al. (2009) Cherepanov and Wackernagel (1995) pEXT22 Protein over-expression vector carrying kanamycin resistance Dykxhoorn et al. (1996) pEXT22/ tatABC pEXT22 expressing TatABC Barrett et al. (2003)  The bacterial strains and plasmids used in this work are listed in Table 1 and oligonucleotides are listed in Supplementary Table S1. Standard methods for cloning and manipulating DNA fragments were used throughout (Sambrook & Russell, 2001). Derivatives of pDOC-K (Lee et al., 2009) and pCP20 (Cherepanov & Wackernagel, 1995) were maintained in host cells using media supplemented with 100 μg ml −1 ampicillin, pACBSR (Herring, Glasner, & Blattner, 2003) was maintained with 30 μg ml −1 chloramphenicol and pEXT22 (Dykxhoorn, St Pierre, & Linn, 1996) derivatives with 50 μg ml −1 kanamycin. All bacteria were cultured in LB medium (Sigma, Gillingham, Dorset, UK).

| Construction of TatExpress strains
To construct the TatExpress series of strains, in which ptac is inserted upstream of the tatA promoter, gene doctoring methodology was used (Lee et al., 2009) (Figure 1). Initially, the ubiB homology region was amplified using PCR with primers UbiBFw and UbiRev and W3110 genomic DNA as FIGURE 1 Construction of the TatExpress strains. (a) The panel shows the construction of the E. coli K-12 TatExpress W3110 strain, using gene doctoring (Lee et al., 2009). Plasmid pDOC-TatExpress was transformed into E. coli W3110 pACBSR and after induction of the SceI meganuclease and λred gene expression (encoded for by pACSBR), the TatExpress gene cassette was transferred onto the W3110 chromosome by homologous recombination. The resultant TatExpress Km1 strain possesses a kanamycin resistance cassette and the ptac promoter upstream of tatA and, thus, tatABCD operon expression is controlled by ptac and the tatA promoter (ptatA). The kanamycin resistance cassette was removed by transforming cells with pCP20, expressing the Flp recombinase, to generate the TatExpress W3110 strain; (b) PCR analysis of the TatExpress W3110 strain. The panel shows an agarose gel of PCR products in which the ubiB-tatA region was amplified from wild-type (WT) E. coli W3110 and TatExpress W3110; (c) Western blot analysis of W3110 TatExpress  SpeI and cloned into pDOC-ubiB, generating pDOC-ubiB/tatA. Finally the ptac promoter from plasmid pEXT22 (Dykxhoorn et al., 1996) was amplified by PCR, using primers ptacXhoI and ptacNdeI and the product was restricted with XhoI and NdeI and cloned into pDOC-ubiB/tatA to generate the gene doctoring plasmid pDOC-TatExpress. This places the ubiB homology region and the ptac-tatA regions between the kanamycin resistance cassette, encoded by the plasmid (Figure 1). The pDOC-TatExpress plasmid was transformed into the E. coli K-12 strain W3110, also carrying the gene doctoring plasmid pACBSR, and gene doctoring was carried out as detailed in (Lee et al., 2009

| Construction of plasmids for protein over-expression
Derivatives of the low copy number plasmid pEXT22 were used for protein over-expression, and the pEXT22/hGH construct was created by overlap extension PCR cloning (Bryksin & Matsumura, 2010). Primers pEXT22Up and pEXT22Down (Supplementary Table S1) were used to PCR amplify the target gene of interest from the pET23 based plasmid described in our previous work (Alanen et al., 2015). The purified PCR product was then used as a template in overlap extension PCR to generate the pEXT22/hGH plasmid described in this study (Table 1). The protein amino acid sequence of the expressed TorA-hGH protein and mature hGH product are detailed in Supplementary Figure S1.

| Sample preparation, protein detection, and quantitation
The preparation of normalized protein samples were carried out as detailed in our previous work (Alanen et al., 2015;Browning et al., 2013).   TatExpress W3110 was considerably higher than wild-type (WT) W3110 ( Figure 1c) and exceeded that produced by W3110 carrying pEXT22/tatABC (Figure 1d, lanes 6 and 7). IPTG-dependent control of TatA expression in TatExpress W3110 was also achieved by providing the lacI q gene, which is carried by the empty pEXT22 vector (Figure 1d, lanes 10 and 11). Similar high-level expression of TatA was also achieved with the equivalent BL21 strain TatExpress BL21 (Supplementary Figure S3).

Growth of both TatExpress derivatives was indistinguishable
from their respective WT strains in shake-flask cultures using standard LB medium (Figure 2). Similarly, the TatExpress strains showed no growth inhibition in tests using a variety of other media (data not shown). Thus, we have engineered a series of industrially relevant E. coli strains to express tatABCD to high levels. Additional experiments identified the optimal IPTG concentration for export of hGH in each strain (data not shown), and the strains' export capacities were tested using a construct in which mature hGH was expressed with an N-terminal TorA signal peptide and a C-terminal hexa-histidine tag (His 6 ) from a pEXT22 based plasmid. Similar tests were carried out using a second target molecule: a single chain antibody fragment (scFv) that has been previously shown to be efficiently exported by Tat if a TorA signal peptide is attached (Alanen et al., 2015). This construct was expressed in WT W3110 and BL21 strains, and in the TatExpress variants (W3110 TE and BL21 TE) and export assays were carried out after 3, 4, and 5 hr induction. The periplasmic fractions were immunoblotted to detect the export scFv ( Figure 5). In some cases the cells exhibit lowered export by the 5 hr time point, which reflects the onset of stress as the cells go into stationary phase, but the data for the 3 and 4 hr time points show a 3.3 | Optimization of induction protocol is a key factor in the high-level export capacity of TatExpress strains A significant point to emerge from these studies is that optimal results are only achieved by matching the synthesis of the substrate to the Tat system's export capacity. This is illustrated by Figure 6, which shows the effects of increasing IPTG concentration from 10 µm to 1 mm on export efficiency and growth characteristics of the TatExpress W3110 strain. Figure 6a shows that, while efficient export of hGH to the periplasm is observed at all of the IPTG concentraions, the lowest concentration of IPTG actually yields the highest level of mature hGH in the periplasm.

| TatExpress strains exhibit enhanced export of hGH and an scFv
We believe that the higher IPTG concentrations do generate more protein product, as would be expected, but that the "excess" hGH is degraded if the Tat system is unable to export it. hGH appears to be particularly susceptible to degradation by cytoplasmic proteases; Supplementary Figure S5 shows that expression of TorA-hGH in a different strain, MC4100, leads to efficient export to the periplasm whereas expression in the Δtat strain leads to an almost complete loss of protein. In contrast, cytoplasmic expression of TorA-scFv is readily apparent in Δtat cells. Figure 6b shows that raising the concentration of FIGURE 3 Tat-dependent export of TorA-hGH in W3110 and TatExpress W3110 cells. Coomassie blue stained 12.5% SDS-PAGE gels showing the export of mature hGH (22 kDa, arrowed) into the periplasm of W3110 wild-type (a) and TatExpress W3110; (b) cells preand post-induction using 10 µm IPTG; (c) Western blot of periplasmic samples from (a) and (b) probed using anti-C-terminal-His 6 antibody. TatExpress W3110 cells show a >5 increase in exported protein at 4 hr post induction relative to export in wild-type cells. Labels are as follows: C, cytoplasmic fraction; M, membrane and insoluble fraction; P, periplasmic fraction; Pre, pre-induction; PI, post-induction. For each lane a normalized amount of protein has been loaded, equivalent to OD 600 0.08 AU total cells (Coomassie blue stained gels), and OD 600 0.008 AU (immunoblots) IPTG also leads to a progressive inhibition of growth, which is consistent with the excess target protein leading to cell stress.

| DISCUSSION
The Tat system's potential as a biotechnological platform has been reported in several studies but low secretion rates have been a serious barrier to industrial exploitation. This platform has clear uses for large scale protein production in fed-batch fermentation systems, and future studies will assess its potential in fed-batch fermentation systems that more closely mimic industrial production processes. However, it should also be noted that even this simple shake-flask system has the ability to

CONFLICTS OF INTEREST
The authors declare no competing financial or other interests.

FIGURE 6
Excessive IPTG concentrations lead to inhibition of growth and reduced export in TatExpress cells. (a) expression of TorA-hGH in W3110 TatExpress cells was induced by the addition of IPTG at the concentrations shown (after 3 hr growth at approximately 0.5 AU). Cells were fractionated to generate cytoplasm/membrane/periplasm samples and the fractions were immunoblotted as shown in Figure 3; (b) effects of IPTG concentration on growth curves of the cultures; uninduced cells are represented by squares and the growth characteristics of cells induced using 10 µm IPTG (diamonds), 100 µm IPTG (triangles), and 1 mm IPTG (crosses) are illustrated