Co-expression for intracellular processing in microbial protein production.

Biotechnol Lett (2014) 36:427–441 DOI 10.1007/s10529-013-1379-z

REVIEW

Co-expression for intracellular processing in microbial protein production Quinn Lu • Juan C. Aon

Received: 12 August 2013 / Accepted: 4 October 2013 / Published online: 16 October 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract The biological activity of a recombinant protein is highly dependent on its biophysical properties including post-translational modifications, solubility, and stability. Production of active recombinant proteins requires careful design of the expression strategy and purification schemes. This is often achieved by proper modification of the target protein during and/or after protein synthesis in the host cells. Such co-translational or post-translational processing of recombinant proteins is typically enabled by coexpressing the required enzymes, folding chaperones, co-factors and/or processing enzymes in the host. Various applications of the co-expression technology in protein production are discussed in this review with representative examples described. Keywords Chaperones Co-production Pathway assembly Post-translational modifications Protein expression

Q. Lu (&) Department of Biological Reagents & Assay Development, Platform Technology & Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA e-mail: [email protected] J. C. Aon Microbial Cell Culture Development, BioPharm R&D, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA e-mail: [email protected]

Introduction Production of biologically active proteins in microbial hosts, especially in Escherichia coli, has been facilitated by the availability of a variety of expression vectors, hosts, and expression strategies. Depending on the source and properties of the protein to be produced, the host cells may need to gear up to meet the needs required for high level production of the protein, which include production of sufficient quantities of endogenous and/or exogenous factors required for high level expression, correct folding, post-translational modifications, and/or processing. All of the above needs can be achieved by co-expression. In addition, assembly of a multi-enzyme complex or an entire pathway in the host cells would simplify downstream procedures and increase scalability. This review summarizes various applications facilitated by the co-expression strategy, in an effort to stimulate further thoughts and enhance utilities in microbial protein production and bioprocess development.

Co-expression for protein folding and translation Co-expression with protein chaperones for protein folding Heterologous protein production in E. coli is often hampered by formation of inclusion bodies which are composed of misfolded and aggregated recombinant

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proteins. This outcome, while protein-specific, can be caused by one or more factors, ranging from rate of transcription and translation, lack of proper modifications/processing, lack of native binding partners or accessory factors, to insufficient expression of folding chaperones. Delineating the causal factor(s) is a challenging task, which may require empirical and/ or systematic investigation. Protein misfolding caused by lack of proper folding assistance can be addressed by co-expression with one or more protein folding chaperones derived from the same or different organisms. A variety of molecular chaperones have been characterized to assist protein folding during and after protein synthesis (Kolaj et al. 2009). These factors include trigger factor that possesses peptidyl-prolyl isomerase activity, hsp70 family members DnaKDnaJ-GrpE, and hsp60 family member GroES and associated GroEL. When co-expressed or fused with the target protein, these chaperones enhance intracellular protein solubility and yield for a variety of target proteins in E. coli (Kolaj et al. 2009; Marco et al. 2007; Kyratsous et al. 2009). For proteins with disulfide bonds, co-expressed thiol-disulfide oxidoreductase DsbC was shown to enhance proper disulfide bond formation in the E. coli periplasm (Depuydt et al. 2011; Berkmen, 2012), or in the cytoplasm of E. coli trxB-gor- mutant strains (Lobstein et al. 2012). Commercially-available plasmid vectors for chaperone co-expression in E. coli are listed in Table 1. While chaperone co-expression offers the potential to enhance the solubility and yield of a recombinant protein, it is not without a consequence on the host cells when the chaperone protein, often a set of such proteins, is also over-expressed. As summarized in a recent report (Martinez-Alonso et al. 2010), such undesired side effects include growth inhibition, reduced yield, proteolysis, reduced protein quality such as low specific activity, and formation of soluble aggregates. It should be pointed out that formation of soluble aggregates can be misleading, since they appear in the soluble fraction upon cell lysis and centrifugal separation (at 12,000–16,0009g), and can often be partially enriched/purified via affinity chromatography. A study on tyrosine kinase production in E. coli revealed that chaperone-enhanced soluble expression for a set of proteins, as observed via IMAC enrichment and SDS-PAGE analysis, were found to be soluble aggregates when analyzed by size exclusion chromatograph (Haacke et al. 2009). It is thus

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Biotechnol Lett (2014) 36:427–441 Table 1 Commercially available vector/host systems for folding chaperones Suppliers

Vectors for chaperones

Strains containing

Notes

TaKaRa (www. takara-bio. com)

pG-KJE8, pGro7, pKJE7, pGTf2, pTf16

BL21

pACYC based vectors with various chaperones and foldases, Camr. For more details, see reference Nishihara et al. (1998)

Shuffles strains, with DsbC

For more details, see reference Lobstein et al. (2012)

trxB- gor(Origami strains)

pET32 expresses H6-TrxA fusion proteins in the cytoplasm of Origami strains

NEB (www.neb. com)

Novagen (www. emdbioscience. com)

pET32

important to include additional analysis of the products, ideally with a functional assay. Co-expression to supply rare codon tRNAs It has long been observed that heterologous protein expression is influenced by differences in codon usage among different organisms. Codons that are rarely used (\1 %) in highly expressed genes in a particular host are referred to as rare codons. Over-expression of a gene with its native sequence may create rare codons in a heterologous host, and depending on the content and locations, these rare codons may cause one or more issues during translation, including frame shift, premature termination, amino acid misincorporation, and in-frame translational hop. Given that codon usage is proportional to the abundance of its cognate tRNA, genes for E. coli rare codon tRNAs have been coexpressed for protein production in E. coli to overcome issues resulting from codon bias. For example, in producing human relaxin 2 in E. coli, co-expressing rare codon tRNAs including arginyl-tRNAArg

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Table 2 Commercially available vector/host systems for rare code tRNAs Suppliers

Vectors for rare codons

Strains containing

Novagen (www.emdbioscience. com)

pRARE, pRARE2, pLysSRARE, pLacIRARE

Rosetta,

Notes

Rosetta (DE3), Rosetta-gami (DE3), RosettaBlue (DE3)

Agilent (www.genomics.agilent. com) Delphi Genetics (www. delphigenetics.com)

BL21-CodonPlus, BL21CodonPlus (DE3) pSCodon1.3, pSCodonGST1.2, pSCherry2, pSCherry4

successfully prevented multiple translational frame shift events associated with rare arginine codons (Kerrigan et al. 2008). Co-expressed rare codon tRNAs also can enhance production of a variety of eukaryotic proteins in E. coli when their native gene sequences are used (Burgess-Brown et al. 2008; Tegel et al. 2010). This strategy provides an option to potentially enhance expression level and/or protein solubility. Plasmid vectors and/or E. coli strains expressing a combination of rare codon tRNAs are commercially available (Table 2). This tRNA co-expression strategy may be effective in cases where abundance of one or more tRNA is an issue but it cannot address issues associated with RNA secondary structures and those that require strategic positioning or engineering of codons. Computer algorithms/programs incorporating various considerations (codon usage, GC content, RNA stability, etc.) have been made available by virtually all gene synthesis companies to enable optimized custom gene design [see for example (Villalobos et al. 2006) and references therein]. Indeed, improved expression has been achieved for a majority of codon-optimized genes heterologously expressed in E. coli (BurgessBrown et al. 2008; Maertens et al. 2010).

Co-expression for protein post-translational modifications (PTMs)

Carstens (2003)

StabyCodon, CherryCodon

considered as an advantage if the recombinant protein is made for crystallization requiring homogeneity. A growing number of PTMs in prokaryotic cells have been uncovered recently, which includes phosphorylation, acetylation, and glycosylation. High throughput methodologies are being developed to further explore the functional PTM sites. All of these studies argue for functional roles of many PTMs in prokaryotes, and their functions being similar to those in eukaryotes. However, while certain PTM substrate sites are conserved from bacteria to mammals, most eukaryotic/mammalian proteins cannot be efficiently and correctly modified when overexpressed in a prokaryotic host such as E. coli. This may largely be due to the extreme evolutionary divergence of PTM enzymes as well as substrates between eukaryotes and prokaryotes, so as to limit the type, extent, and efficiency of the reactions to a level beyond practical uses. In addition, even in cases when the host system possesses a functional PTM enzyme, such as when a yeast host is used, the biological levels of the enzyme may not be sufficient to modify the overly expressed substrate protein. For producing proteins with a desired PTM, a reasonable practice is to supply the specific PTM enzyme via co-expression. Examples of some types of PTMs enabled by the strategy can be found in Table 3. This section discusses such efforts for three selected reaction types. Co-expression for protein acetylation

Historically, prokaryotic cells are viewed as lacking many types of protein PTMs commonly found in eukaryotic hosts, and are thus considered to be disadvantageous for use as a host for producing recombinant proteins where a post-translational modifications (PTM) is essential. However, this is often

Gram-negative bacteria, such as E. coli, are capable of acetylating components of their own proteome; however, it appears to occur infrequently and is undertaken by a discrete molecular pathway from that in eukaryotic cells. Three E. coli N-a-acetyltransferases

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Table 3 Examples of PTMs in microbial hosts enabled by co-expression PTM

Substrate

Coexpressed enzyme

Comment

Reference

Phosphorylation

CREB kinase inducible domain

PKA

Phosphorylation

ASF/SF2

SRPK1

Phosphorylation

p53

GCN5

Phosphorylation

JDP2, ATF2

JNK1

Murata et al. (2008)

Phosphorylation

Histon H3 tail

Phosphorylation

Hsp90, Grp94

Aurora kinase B Casein Kinase II

Murata et al. (2008) Shi et al. (1994)

Phosphorylation

HSP20

PKG

Flynn et al. (2007)

Phosphorylation ? acetylation

Histone H3 tail

Aurora kinase B ?PCAF

PCAF = histone acetyltransferase

Murata et al. (2008)

Dephosphorylation

Hck

YopH

Hck is a Src family kinase, YopH is a phosphotyrosine phosphatase

Kristelly et al. (2011)

Dephosphorylation

c-Abl, c-Src

YopH

YopH is a phosphotyrosine phosphatase

Seeliger et al. (2005)

Na-Acetylation

Thymosin a1

RimJ

NAT

Ren et al. (2011)

Myristoylation

NMT

Click chemistry

Heal et al. (2012)

Myristoylation

MAP, NMT

MAP to remove Met, and NMT to

Valkenburgh and Kahn (2002)

NCS = neuronal calcium sensor

De Cotiis et al. (2008)

Sugase et al. (2008) Phosphorylated ASF/SF2 increased solubility and activity Expressed as GST-p53-GCN5 fusion

Yue et al. (2000) Acharya et al. (2005)

Myristoylation

NCS-1

NMT

Hydroxylation

HIF1a C-terminal activation domain

FIH

Biotinylation

BP-Ubiquitin, BPSUMO, BP-UBP41

birA

BP = Biotinylation peptide (MASSLRQILDSQKMEWRSNAGG)

Wang et al. (2003)

Methylation

Sbp1, Stm1

HMT1

Type I Arginine methyltransferase PRMT

Hsieh et al. (2007)

Sumoylation

RanGAP1, RanBP2, p53, TONAS-ring, PML

SUMO-1 or SUMO-2

SUMO-E1 (Aos1-Uba2) and SUMO-E2 (Ubc9) also co-expressed

Uchimura et al. (2004a, b)

Sumoylation

MBD1

SUMO-1 or SUMO-3

SUMO-E1 (Aos1-Uba2) and SUMO-E2 (Ubc9) also co-expressed

Saitoh et al. (2009)

Phosphopantetheinylation

NRPS module PheAT

Gsp

Gsp, a PPTase from Bacillus brevis

Ku et al. (1997)

Phosphopantetheinylation

Polyketide synthases (PKS), Nonribosal peptide synthetases (NRPS)

Sfp

Sfp, a PPTase from Bacillus subtilis

Sunbul et al. (2009)

Gluconoylation

Liver X receptor, elongin C

Pgl

Removal of gluconoylation and/or phosphogluconoylation

Aon et al. (2008)

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Sugase et al. (2008)


(NATs), RimL, RimJ, and RimI, have been identified and each appears to work on a limited set of substrate proteins (Vetting et al. 2005; Miao et al. 2007). This is in contrast to the relatively wider range of substrate specificity observed with eukaryotic NATs (Polevoda et al. 2009). A few proteins expressed in E. coli are Na-acetylated (Wu et al. 2006; Bariola et al. 2007), however, the reactions were mostly partial and further engineering would be required for efficiency. For example, when a fusion protein of thymosin a1 and L12, (thymosin a1-L12), is expressed in E. coli, the Nterminus of thymosin a1 was found to be partly N-aacetylated by an endogeneous enzyme RimJ, an full acetylation of the fusion protein was achieved via coexpression with RimJ (Ren et al. 2011). Up to 98 % of eukaryotic proteins are N-terminally acetylated, and the reactions are thought to take place co-translationally at the ribosome (Arnesen et al. 2009). Six different classes of NAT complexes in eukaryotes, NatA-NatF, have been described, each composed of distinct catalytic and regulatory subunits (Polevoda et al. 2009; Arnesen 2011). Substrate specificities of these NATs appear to be dependent mostly on the first 2–8 amino acid residues at the Nterminus. For examples, NatA acetylates N-termini with Ser, Ala, Gly, Thr, or Val after removal of Met; whereas other NATs acetylate the initiator Met with specificities depending on penultimate residue. Johnson et al. (2010) successfully expressed a few acetylated eukaryotic proteins in E. coli by co-expressing the fission yeast NatB acetylation complex Naa20p/ Naa25p. Three of the four target proteins expressed (SkTm, Cdc8, Tfs1) showed a two to threefold increase in yield in comparison to standard expression systems. Aside from N-terminal acetylation, protein acetylation at internal sites has also been achieved via co-expression. p53 acetylated at Lys320 was produced in E. coli when fused with Gcn5, a yeast homologue of human PCAF (Acharya et al. 2005). The N-terminal tail of histone H3 was both acetylated and phosphorylated via co-expression with PCAF and Aurora kinase B (Murata et al. 2008).

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catalyze the reaction for most of the substrates of mammalian origin. Other than those that can be autophosphorylated, many of the mammalian kinases expressed in E. coli are unphosphorylated and their activity impaired. Indeed, many protein kinases for which the crystal structure has been solved so far have been crystallized in an inactive state; significant structural differences have been observed between active and inactive forms of some kinases (Johnson et al. 1996). A number of phosphorylated mammalian proteins have been produced in E. coli via co-expression with a corresponding kinase of eukaryotic origin (Table 3). For example, heat shock protein hsp20 and Hsp90 were phosphorylated via co-expression with PKG and casein kinase, respectively (Flynn et al. 2007; Shi et al. 1994). In the case of hsp90, the co-expressed casein kinase II specifically phosphorylated recombinant human and yeast hsp90 and a closely related protein Grp94 from bovine. In expressing ASF/SF2, a mammalian pre-mRNA slicing factor, Yue et al. (2000) reported that co-expression in E. coli with its kinase SRPK1 significantly enhanced solubility of ASF/SF2, and the phosphorylated protein was more active than the unphosphorylated form. An observation with kinase expression in E. coli is a low yield associated with the toxic effect of the produced kinases, especially for those that are autophosphorylated upon synthesis, such as cAMP-dependent kinase and Src tyrosine kinases (Wang et al. 2006; Kemble et al. 2006). A strategy that is well established to circumvent this issue is to produce the protein as an unphosphorylated form via co-expression with a phosphatase. After purification, the product can then be activated by auto-phosphorylation in the presence of Mg2?/ATP. The approach was successfully applied to the receptor tyrosine kinases c-Abl, c-Src, and Hck (Wang et al. 2006; Seeliger et al. 2005; Kristelly et al. 2011). In the above cases, significantly enhanced yields were achieved by kinase/phosphatase co-expression either as a single transcript via direct fusion (Wang et al. 2006) or as two separate transcripts (Seeliger et al. 2005; Kristelly et al. 2011).

Co-expression for protein phosphorylation Phosphorylation is a post-translational modification at Ser/Thr/Tyr that is required for activity of a variety of enzymes including kinases. Given the evolutionary distance, E. coli endogenous enzymes are unable to

Co-expression to eliminate phosphogluconoylation Phosphogluconoylation and/or gluconoylation are PTMs dependent on the formation of 6-phosphoglucono-1,5-

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lactone (6-PGLac) which is an intermediate of the pentose phosphate pathway produced by glucose-6phosphate dehydrogenase. 6-PGLac is involved in nonenzymatic glycation reactions in vivo. Protein glycation can reduce enzyme activity of a protein (Beranek et al. 2001) or interfere with protein crystallization (Kim et al. 2001). Furthermore, spontaneous a-N-6-phosphogluconoylation modifications have been observed when heterologous proteins are expressed in E. coli (Mironova et al. 2003), or when hexahistidine fusion proteins are expressed (Yan et al. 1999). In high-cell density microbial cultures, 6-PGLac, gluconolactone (the dephosphorylated form of 6-PGLac), and glyceraldehydes are accumulated and can become potent agents for covalent modification of proteins in eukaryotes and prokaryotes (Rakitzis and Papandreou 1998). Under typical E. coli growth conditions, 6-PGLac is hydrolyzed to 6-phosphogluconic acid with the action of phosphogluconolactonase enzyme, Pgl (EC 3.1.1.31). E. coli strain BL21(DE3) possesses very low endogenous Pgl activity (Wang et al. 2005). This may provide an explanation for why some recombinant proteins expressed in the B strain are phosphogluconoylated and/or gluconoylated, as observed in our laboratories as well as reported in the literature. Follow-up studies in our laboratories indicate that the PTMs can be suppressed when the recombinant protein is expressed in a K-12 strain. For example, expression of an 18 kDa protein in a K-12 strain MG1655 showed no detectable levels of gluconoylated product, whereas a gluconoylated product was evident when the same protein (contained in the same expression plasmid) is expressed in a B strain BL21(DE3) (Fig. 1a, b). Based on these observations, the accumulation of 6-PGLac and the formation of aN-phosphogluconoylated and/or gluconoylated products in E. coli B strains was postulated to arise from the absence of Pgl activity (Wang et al. 2005; Aon et al. 2008). Aon et al. (2008) further demonstrated that protein phosphogluconoylation and/or gluconoylation in B strains can be suppressed via co-expression with a pgl gene from Pseudomonas aeruginosa (Fig. 1c). This strategy was further applied to two other proteins to eliminate phosphogluconoylation and/or gluconoylation adducts when expressed in E. coli BL21(DE3), including the liver X receptor and elongin C. The general applicability of these findings could have a significant economic impact on commercial production of heterologous proteins in E. coli.

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Co-expression with engineered tRNA for amino acid specific modification Protein modifications at any defined amino acid residue can be achieved via site-specific incorporation of unnatural amino acids (UAA). This is achieved by tRNA engineering which involves the use of a mutant tRNA that suppresses/recognizes a blank codon such as the amber codon TAG, and evolution and selection of its cognate aminoacyl-tRNA synthetase (aaRS) that uniquely recognizes a UAA (UAARS) (Liu and Schultz 2010). A variety of such UAARS/tRNA pairs has been developed and used to incorporate/encode corresponding UAAs into specific sites in recombinant proteins via co-expression. These modifications range from incorporating naturally occurring PTMs that are typically installed enzymatically to unnatural amino acid derivatives that are challenging to achieve with the common 20 amino acids (Liu et al. 2009; Neumann et al. 2008), including metal ion-binding, photoisomerizable, photocaged, and photoreactive amino acids (Hendrickson et al. 2004; Liu and Schultz 2010). For example, recombinant rat manganesesuperoxide dismutase (MnSOD) was produced in E. coli with site-directed incorporation of Ne-acetyllysine at position 44 and 3-nitrotyrosine at position 34, respectively, by co-expressing with specific orthogonal UAARS/tRNA pairs (Neumann et al. 2008). Nitration of Y34 abolished the catalytic activity of MnSOD. The ability to modify amino acid residues site-specifically adds a new dimension to protein engineering and evolution, including modulation of biophysical and biochemical properties (solubility, stability, activity, immunoreactivity) of recombinant proteins or antibodies for protein functional studies and therapeutic applications.

Co-expression for intracellular processing Expression of recombinant proteins as protein fusions is a popular strategy to ensure proper translation, folding, and to facilitate purification. Separation of fusion proteins can be achieved at various stages either by co-translational protease processing inside the host cells or by protease cleavage after cell lysis and/or protein purification. Co-translational processing occurs shortly after protein synthesis by an endogeneous protease or by a co-expressed protease. Co-


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Fig. 1 Pgl activity is required to remove the gluconoylated variant formation in an 18 kDa protein. Both samples from end of fermentation were collected, and analyzed by the reversed phase HPLC of whole cell lysate from Fed-Batch cultures of the K-12 strain (panel A), E. coli BL21 (DE3) strain (panel B), and

E. coli BL21 (DE3) ? pgl strain (panel C). Main peak was the native 18 kDa protein and peak in the circle corresponded to the gluconoylated variant. Panel C is the overlay of HPLC profiles from E. coli BL21 (DE3) strain (blue) and E. coli BL21 (DE3) ? pgl strain (purple)

translational processing ensures production of proteins with desired PTMs for their proper activities.

the addition of N-alpha acetyl groups by methionine aminopeptidases (MAP) and N-a-acetyl transferases, respectively. These N-terminal amino acid residues and their modifications are important for protein activity and/or stability. For protein production in E. coli, the processing protease can either be an endogenous enzyme or be supplied via co-expression. Two strategies have been commonly utilized to achieve the goal: the MAP processing approach and the ubiquitin processing approach. The processing enzymes for these approaches can be supplied via coexpression. The MAP processing approach involves addition of an AUG codon to the ORF for the mature peptide for expression as a methionylated peptide. The initiator Met can then be removed in E. coli by the endogenous MAP or co-expressed MAP. For example, an initiator Met added to the mature peptides of human adult globin HbA (a and b subunits) was efficiently removed by a co-expressed E. coli MAP (Shen et al. 1997). However, without the overexpressed MAP, the methionylated a and b subunits were mostly unprocessed (Shen et al. 1993). Early studies indicated that the cleavage efficiency of the E. coli MAP is limited by the side-chain radius of the penultimate amino acid

Intracellular processing for protein activation Many classes of active proteins are synthesized as proproteins and activated by a protease based on a physiological condition. To produce active proteins in E. coli, the activating enzyme required for processing may need to be supplied via co-expression. For example, an engineered vibriolysin gene was coexpressed to activate an aminopeptidase expressed in E. coli by removing the propeptide of the enzyme (Sonoda et al. 2009), which is inhibitory to its activity but required for proper folding. Another example is IL-18, where Pro-IL18 was co-expressed with caspase 1 (ICE) or caspase 4 for intracellular activation (Kirkpatrick et al. 2003). Intracellular processing to produce proteins with authentic N-terminal residue Natural N-terminal processing of proteins in eukaryotes occur both co- and post-translationally, which involves removal of the initiator methionine (Met) and

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residue, the amino acid residue following Met (Hirel et al. 1989), with higher efficiency when the penultimate residue is small. This issue has been addressed by using engineered MAPs with improved substrate acceptance. For example MAP with Y168G/M206T/Q233G is able to remove the Met from recombinant proteins with bulky or acidic penultimate residues (Liao et al. 2004). Efficient MAP removal also set the stage for additional modification of the penultimate residue. In producing N-terminal myristoylation of human ADP-ribosylation factor family GTPases in E. coli, that both a MAP and an N-myristoyltransferase (NMT) were required for efficient modification of the recombinant proteins, with MAP for Met removal and NMT for acylation (Valkenburgh and Kahn 2002). Along the same line, co-expression with peptide deformylase enhanced deformylation of N-terminal methionine of a recombinant protein in E. coli (Warren et al. 1996), demonstrating an added approach in preparing the Ntermini of recombinant proteins for MAP-mediated Met removal (Solbiati et al. 1999). Overall, this Met processing approach has been established as a platform to produce proteins with authentic N-terminal amino acid residues, although in certain cases optimization of expression conditions may be needed to maximize MAP processing in E. coli (Covalt et al. 2005). The ubiquitin processing approach involves expression of the passenger protein with an N-terminal ubiquitin tag, which is then removed via intracellular processing. Ubiquitin is used for such a design since ubiquitin moiety can be removed via an ubiquitinspecific protease by cleaving specifically at the Cterminus of ubiquitin, without leaving undesired residues on the passenger protein. Given that prokaryotic cells lack ubiquitin/protease system, ubiquitinspecific proteases have been co-expressed to remove ubiquitin tag from fusions expressed in E. coli. An added advantage of the system is that translational fusions with ubiquitin or small ubiquitin-like modifier protein (SUMO) are thought to increase translation and folding efficiency of the passenger protein (Baker et al. 2005). The proper folding of the moiety and/or its fusion proteins is required for proper cleavage of the ubiquitin/SUMO moiety. Ubiquitin-based intracellular processing has been used to produce a number of proteins in E. coli. For example, Ubp1 was coexpressed in E. coli to remove the ubiquitin tag from a

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fusion with RNA-dependent RNA polymerase 3Dpol (Ub-3Dpol-H6) (Gohara et al. 1999), human growth hormone (Ub-hGH), and IFNa(Ub-IFNa) (Zaleski et al. 2012). In the cases for hGH and IFNa, Ubp1 was expressed from a novel vector pIGPZ bearing an origin of replication that is compatible with other vectors bearing ColE1, p15A, or pSC101. Importantly, co-existence of pIGPZ with another plasmid bearing ColE1, p15A, or pSC101 can be maintained over multiple passages in the absence of antibiotics selection (Zaleski et al. 2012). This feature of the coexpression system would enable intracellular processing at scale. [Note that in all of the cases discussed above, the ubiquitin/SUMO fusion proteins can alternatively be purified first and the tag removed in vitro with a purified Ubp (Baker et al. 2005; Lu et al. 2009).] Intracellular processing to remove translational fusion partners A variety of protein tags have been utilized to enhance solubility via direct fusion, including MBP, Trx, NusA, SUMO, and a set of highly acidic proteins from E. coli. These solubility tags were proposed to have intramolecular chaperone activity, which promotes proper folding upon synthesis of their respective fusion partners (Kapust and Waugh 1999; Nallamsetty and Waugh 2006; Jurado et al. 2006; Varshavsky, 2005; Malakhov et al. 2004). The fusion proteins are typically purified and processed in vitro to remove the tag. However, certain passenger proteins tend to form aggregates after tag removal. Intracellular processing would allow early detection of such fusion constructs, in addition to eliminate the tag removal step. It thus provides a cost-effective way to produce recombinant proteins in large scale. The site-specific protease TEV has been demonstrated to work in E. coli. In the cases studied, TEV was co-expressed with ‘‘MBP-tevpassenger-His6’’ fusions to remove the MBP tag intracellularly (Kapust and Waugh 2000). This technique has been demonstrated to work efficiently for a variety of passenger proteins (Kapust and Waugh 2000; Austin et al. 2009; Nallamsetty and Waugh 2006; Shih et al. 2005). To prolong MBP association, hence chance of proper folding, induction of TEV expression was delayed by 2 h from induction of the MBP/passenger fusions, which led to enhanced solubility for two passenger proteins out of four tested (Kapust and Waugh 2000). After intracellular


processing, the C-terminally His6 tagged passenger proteins can then be purified via Ni–NTA chromatography. Similarly, the protease from tobacco vein mottling virus (TVMV) was successfully used to process MBP-TVMV-passenger fusion proteins intracellularly (Donnelly et al. 2006).

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5-aminolevulinic acid, providing a cost-effective alternative for P450 production at scale. Another example is the production of active NADP-dependent [NiFe]-hydrogenase, SHI (Sun et al. 2010). In the study, in addition to four structural genes for SHI, up to eight processing genes (hypCDABEF, hycI, hlyD) and one NADPH reductase gene ferredoxin oxidoreductase A (frxA) were co-expressed.

Co-expression for pathway assembly Given the heterologous nature, optimal levels of expression and activity of recombinant proteins in E. coli may require presence of its native cofactors, in addition to those involved in direct modification of the target protein. Such factors may include its native binding partners for complex formation, those involved in redox regeneration, or those involved in biosynthesis of a cofactor. In some cases, proteins from the entire pathway may need to be co-expressed. Examples of co-expressing physically interacting proteins for complex formation can be found in a recent review (Kerrigan et al. 2011), elaborated below are selected cases where protein activity is significantly enhanced by the co-expression of other auxiliary factors. Co-expression of cofactors required for protein activity Production of active P450 enzymes in E. coli requires an efficient system to enable proper electron transfer to the heme iron in P450. This has been achieved via coexpression with a redox partner(s), such as NAD(P)HP450 oxidoreductase, ferredoxin (fdx)/fdx-oxidoreductase, or flavodoxin (fld)/fld-oxidoreductase (Hlavica 2009). For example, the activity of human P450cl7 expressed in E. coli was increased by 100-fold upon co-expression with the rat NADPH cytochrome P450 oxidoreductase (Ehmer et al. 2000). In addition to redox partners, a sufficient level of free heme is also required for incorporation into the recombinant P450 protein. This is usually achieved by supplementing the medium with 5-aminolevulinic acid. However, Harnastai et al. (2006) harnessed the E. coli heme biosynthesis pathway by over-expressing the glutamyl-tRNA reductase (hemA) gene, which catalyzes a rate limiting step in heme production. In their study, microsomal and mitochondrial CYPs were produced to a level that is equivalent to or higher than using

Co-expression of pathway enzymes to produce cyclic peptides Lantibiotics are a class of ribosomally synthesized natural products that possesses antimicrobial activities. They are cyclic peptides containing unusual amino acid residues that result from PTM of a prepeptide. Upon synthesis of the prepeptide, specific serine and threonine residues are dehydrated to dehydroalanine (Dha) and dehydrobutyrine (Dhb) residues, followed by cyclization of Dha or Dhb residues with the thiol group of a specific cysteine residue to form lanthionine (Lan) or 3-methyllanthionine (MeLan), respectively. The modified peptide is then exported and the N-term leader peptide removed via a protease action. Biosynthesis of a number of lantipeptides in E. coli have employed a strategy where amino acid modifications (dehydration and cyclization) are achieved intracellularly via co-expression with specific modifying enzymes, followed by purification of the modified precursor peptide and removal of the leader peptide in vitro (Shi et al. 2011; Nagao et al. 2005; Lin et al. 2011). This ‘‘leader-on’’ strategy avoids potential cytotoxic effects of the mature peptides to the expression host (Valsesia et al. 2007). In producing lantipeptide nisin in E. coli, nisA/nisB/nisC were co-expressed, in which nisA encodes for the prepeptide and is tagged with hexaHis (H6-NisA), nisB for dehydration, and nisC for cyclization (Shi et al. 2011). Upon purification of the modified fusion protein H6-NisA, the leader peptide was then removed by treating with trypsin. Lantinized nukacin ISK-1 was similarly produced in E. coli via co-expressing nukA/nukM, where nukA encodes the prepeptide and nukM encodes an enzyme with both dehydratase and cyclase activities (Nagao et al. 2005). In activating lantipeptides ProcA and HalA after biosynthesis in E. coli, efficient removal of the leader peptide in vitro was facilitated by engineering a TEV cleavage site (ENLYFQ) and a Factor Xa

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cleavage site (IEGR), respectively, between the leader sequence and the core peptide (Shi et al. 2011). Peptide cyclization has been applied to producing non-lantibiotic peptides of therapeutic potential for enhanced stability and/or resistance to proteolytic degradation (Moll et al. 2010). By employing the Lactococcus lactis nisin biosynthesis and exporting pathway enzymes nisA/nisB/nisC/nisT, a number of non-lantibiotic peptides were produced in L. lactis to form thioether-cyclized products, including vasopressin, enkephalin, erythropoietin, angiotensin, and lutenizing hormone release hormone LHRH (Rink et al. 2010; Kluskens et al. 2005; Kluskens et al. 2009). Production of these peptides was achieved by coexpressing the installing and exporting enzymes NisB/ NisC/NisT with an engineered NisA fusion protein in which the NisA leader peptide is fused to the target peptide containing designed Ser/Thr and Cys residues at appropriate positions. [Note that NisT is a transporter protein that is responsible for export of prenisin or nisin fusion proteins (Kuipers et al. 2004)]. In both cases for angiotensin and LHRH, thioether-cyclized products demonstrated significantly improved resistance against proteolytic degradation (Kluskens et al. 2009; Rink et al. 2010). Further in vivo studies revealed that a thioether-cyclized angiotensin is significantly more stable than the natural peptide, which led to an enhanced potency in vasodilation by tenfold (Kluskens et al. 2009). Thioether-cyclization of peptide ligands for d-opioid receptor and somatostatin receptor resulted in enhanced selectivity and/or specific activity in receptor binding (Svensson et al. 2003; Osapay et al. 1997). Lan engineering and biosynthesis technology thus provide an added dimension in the designing and production of peptide therapeutics. In cell enzyme immobilization for pathway assembly To enable affinity-mediated purification of recombinant proteins/enzymes at a scale, an in vivo protein immobilization system has been developed, in which the target protein/enzyme is attached to polyhydroxyalkanoate (PHA) granules in a microbial expression host. Enzyme-coated PHA granules can be easily recovered via simple centrifugation steps (Banki et al. 2005; Barnard et al. 2005). With the system, a bacteria-derived operon responsible for the

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biosynthesis of PHAs is expressed from one plasmid, and the target protein is expressed from another plasmid with a phasin tag, which directs deposition of the fusion protein to PHA granules. Importantly, PHAdisplayed proteins remain active, so can be used directly as immobilized enzymes. For example, PHA granules carrying an insect-specific toxin were shown to be active in pest control (Moldes et al. 2006). Further, if desired, active proteins can be released from PHA granules, which can be achieved either via a biophysical treatment or via induced enzymatic autocleavage with an engineered intein (Banki et al. 2005; Barnard et al. 2005). Steinmann et al. (2010) have developed a system for protein display on inclusion bodies in E. coli. With this system, inclusion bodies composed of PhaC tagged with a stretch of negatively charged coil were used as nano-beads, and a galactose oxidase tagged with a positively charged coil was co-expressed and deposited onto the nanobeads via coiled-coil interaction. Functional analysis of the nano-beads indicated that the displayed oxidase remains active. These studies indicated that the in vivo protein immobilization systems provide an attractive option for cost-effective production of enzymes/proteins at scale. Enzymes involved in a pathway have also been engineered to form artificial enzyme scaffolds in E. coli to increase effective enzyme concentration in cells (Dueber et al. 2009). This is achieved by coexpression and assembly of specially designed scaffold docking molecules and component enzyme molecules. The scaffold docking molecule is a tethered fusion protein harboring modular protein–protein interaction domains (i.e., GBD, SH3, and PDZ into a GxSyPz tethered fusion protein where x, y and z represent the numbers of repeats), and the component enzyme molecules are enzymes tagged with a cognate peptide ligand specific for a protein domain in the scaffold docking molecule. For assembly of a specific multi-enzyme scaffold, the stoichiometry of component enzymes can be modulated by varying the number of the corresponding domain in the docking molecule. Using the system, enzyme scaffolds containing three enzymes involved in the synthesis of mevalonate, AtoB, HMGS, and HMGR, were coexpressed and targeted to GxSyPz, respectively, and tested for production titers. A scaffold with G1S2P2 77-fold higher in product titer than the unscaffolded but otherwise co-expressed enzymes (Dueber et al.


2009). The same approach was also applied to increase production of glucaric acid in E. coli. Three enzymes, Udh, Ino1 and MIOX, were co-expressed, targeted to GxSyPz, respectively, and tested for product titer (Moon et al. 2010). Out of the strains with 9 scaffolds tested (where x = 1, y and z are 1, 2, and 4), the scaffold formed with G1S4P4 had a fivefold higher product titer over unscaffolded enzymes. The engineered enzyme scaffold approach thus provides a means to increase enzyme efficiency of a reconstructed pathway in microbial cells. This approach is similar to the polyketide synthase pathways, in which functional modules are linked and/or contained in a complex. An important requirement for the scaffold approach to work may be that at least a fraction of all the designed docking and component molecules synthesized should remain intact and soluble before scaffold assembly, and that the metabolic burden imposed by the recombinant proteins is tolerated by the host to ensure robustness of the system.

Concluding remarks Heterologous protein production in microbial hosts provides a convenient and cost-effective way to produce recombinant proteins ranging from laboratory scale to industrial manufacture. Many of the protein/ peptide/mAb drugs on the market are produced in microbial systems including E. coli and yeasts. However, depending on the origin of the target protein, capability of proper folding in the host cells, and PTMs required for solubility, stability, and activity, additional auxiliary factors and/or processing enzymes may be required. Even in cases where an endogenous factor/enzyme can perform the required action, given the nature of overexpression of the target protein, additional levels of the factor/enzyme may need to be supplemented. As discussed in this article, these auxiliary factors and processing enzymes, heterologous or homologous to the host cells, can be achieved by co-expression. Details of the strategies to achieve protein co-expression in E. coli can be found in a recent review (Kerrigan et al. 2011). Typically, a fit-for-purpose procedure may require engineering and factorial testing of the expression vector(s) bearing various auxiliary factors, the host cell(s), culturing conditions, together with a purification procedure. High-throughput approaches (Peleg and Unger 2008;

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Mehlin et al. 2006) may help reach a fit-for-purpose strategy and process sooner. These initial steps are usually sufficient to allow production of proteins at milligram to gram quantities at a laboratory scale, but further engineering and process development/optimization are required for cost effective and sustainable protein production at scale (Choi et al. 2006). Given the genetic amenability of the microbial hosts, all of the factors required for folding, modification, and/or processing of the target protein can be incorporated as part of the host cell engineering. In other words, any of the co-expressed genes carried on a plasmid (or plasmids) can be pre-transformed into the host cells or even be incorporated into the genome to make specialpurpose strains. Further, the principles discussed can be applied readily to engineer coordinated expression of multiple active enzymes or an entire pathway in a production host. Further exploration in the area would greatly enable pathway engineering and metabolic engineering in a scalable system for a variety of applications, including therapeutic protein production, biocatalysis of chemical reactions, biofuel production, and waste treatment. Acknowledgments We thank Edward Appelbaum, Robert Ames, Angela Bridges, Andrew Fosberry, Murray Brown, and James Fornwald for critical reading of the manuscript. Support from authors’ line managers at GlaxoSmithKline is greatly appreciated.

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Fish processing wastes for microbial enzyme production: a review.

Coexpression of cellulases in Pichia pastoris as a self-processing protein fusion.

Maximizing efficiency of rumen microbial protein production.

Saccharification behavior of cellulose acetate during enzymatic processing for microbial ethanol production.

Protein content and amino acid composition of certain fungi evaluated for microbial protein production.

Escherichia coli BL21(DE3) chromosome-based controlled intracellular processing system for fusion protein separation.

Enhanced membrane protein expression by engineering increased intracellular membrane production.

Microbial biotechnologies for potable water production.

Microbial granulation for lactic acid production.

A temperature-sensitive mutant of Newcastle disease virus defective in intracellular processing of fusion protein.

Combining microbial cultures for efficient production of electricity from butyrate in a microbial electrochemical cell.

Selective Targeting of Cells via Bispecific Molecules That Exploit Coexpression of Two Intracellular Proteins.

Intracellular processing of the porcine coronavirus transmissible gastroenteritis virus spike protein expressed by recombinant vaccinia virus.

Development of protein mimics for intracellular delivery.

Microbial production of 4-thiouridine.

Biotechnological Processes in Microbial Amylase Production.

Advances and bottlenecks in microbial hydrogen production.

Efficiency of microbial growth in biomass production.

Integrated study of fermentation and downstream processing in a miniplant significantly improved the microbial 1,3-propanediol production from raw glycerol.

Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir.

Engineering Escherichia coli for Microbial Production of Butanone.

Cell immobilization for microbial production of 1,3-propanediol.

Microbial fuel cells and microbial electrolysis cells for the production of bioelectricity and biomaterials.

Tyrosine Binding Protein Sites Regulate the Intracellular Trafficking and Processing of Amyloid Precursor Protein through a Novel Lysosome-Directed Pathway.