Recombineering for Genetic Engineering of Natural Product Biosynthetic Pathways

TIBTEC 1876 No. of Pages 14

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Review

Recombineering for Genetic Engineering of Natural Product Biosynthetic Pathways Muhammad Nazeer Abbasi

,1,2,* Jun Fu,1 Xiaoying Bian,1 Hailong Wang,1,* Youming Zhang,1,* and Aiying Li1,*

Microbial genomes encode many cryptic and uncharacterized biosynthetic gene clusters (BGCs). Exploiting this unexplored genetic wealth to discover microbial novel natural products (NPs) remains a challenging issue. We review homologous recombination (HR)-based recombineering, mediated by the recombinases RecE/RecT from Rac prophage and Redα/Redβ from lambda phage, which has developed into a highly inclusive tool for direct cloning of large DNA up to 100 kb, seamless mutation, multifragment assembly, and heterologous expression of microbial NP BGCs. Its utilization in the refactoring, engineering, and functional expression of long BGCs for NP biosynthesis makes it easy to elucidate NP-producing potential in microbes. This review also highlights various applications of recombineering in NP-derived drug discovery.

Highlights Genome mining for the discovery of novel natural products could meet the demand for new anti-infective drugs against drug-resistant pathogens. There are plentiful cryptic and uncharacterized gene clusters of various sizes ranging from 10 to 200 kb that are responsible for the biosynthesis of bioactive compounds, but accessing them remains challenging. Red/ET recombineering, based on homologous recombination, is mediated by recombinases from phage or prophage. It can be used for the refactoring, engineering, and functional expression of long biosynthetic gene clusters.

Contemporary Approaches for Microbial NP Research Elevated drug resistance (see Glossary) in pathogens brings about serious human health challenges, making it necessary to discover new anti-infective drugs [1] (https://www.who. int/drugresistance/documents/surveillancereport/en/). NPs from microbes and their semisynthetic derivatives are a promising source of pharmaceutically important bioactive compounds (Figure 1) [2,3]. Their highly diverse chemical structures facilitate a broad spectrum of bioactivities, including antibiotic, insecticide, and antitumor properties. However, new and effective methods for the discovery of novel NPs from microorganisms are increasingly sought. Conventional drug discovery methods, typically involving bioactivity-based screening of microbial crude extracts, co-culturing and in situ cultivation, have been used to discover NPs, but they have limited efficiency and cannot meet the increasing demand for bioactive NPs against drug-resistant pathogens, resulting in a severe scarcity of potent anti-infective agents. Therefore, there is also an increasing demand for new and effective methods for discovering novel NPs. With the advent of next-generation sequencing (NGS), a huge number of microbial genome sequences are available in public databases, and by using new computational biology tools such as Antibiotics & Secondary Metabolite Analysis SHell (antiSMASH) and Pep2Path, BGCs of NPs can be predicted in batches [4,5]. In recent years, genome mining has emerged as an effective method and has frequently been used for NP discovery. Genome sequencing data has shown that microorganisms have a far greater potential to produce bioactive NPs than has so far been reported [6]. More interestingly, recent reports have indicated that the human microflora contains a large number of BGCs of potential significance to both human health and disease [7,8]. Many of these cryptic or uncharacterized BGCs are considered abundant but untapped resources for the discovery of new and novel biologically active NPs [9–11]. Despite the availability of this huge amount of sequencing data for uncharacterized BGCs in public databases, unfortunately only a few of these BGCs have been characterized so far. This Trends in Biotechnology, Month 2019, Vol. xx, No. xx

A widely applicable tool based on Red/ ET recombineering can be used for direct cloning of large DNA up to 106 kb, seamless mutation, multifragment assembly, and heterologous expression of microbial NP BGCs.

1

Helmholtz International Lab for Anti-Infectives, Shandong University– Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, PR China 2 http://www.shib.sdu.edu.cn/en/Home.htm

*Correspondence: [email protected] (M.N. Abbasi), [email protected] (H. Wang), [email protected] (Y. Zhang), and [email protected] (A. Li).

https://doi.org/10.1016/j.tibtech.2019.12.018 © 2019 Elsevier Ltd. All rights reserved.

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Glossary

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Figure 1. Diversity of Natural Products Produced from Microbes, Including Antibiotics and Antiparasitic and Antitumor Agents. These compounds are representative of natural products isolated from different microorganisms. They display high diversity in structure and bioactivity and their biosynthetic genes are clustered in various sizes on the genome of their native hosts.

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Biosynthetic gene cluster (BGC): a physically clustered group of two or more genes in a particular genome that together encode a biosynthetic pathway for the production of a specialized metabolite (including its chemical variants). Cosmid library: a type of genomic library that is constructed using a cosmid (cos site + plasmid = cosmid) as a vector. The DNA sequence of the cos site is originally from the lambda phage and can be recognized by lambda packaging proteins; it also determines the size range of inserts to be cloned during genomic library construction. Drug resistance: the reduction in effectiveness of a medication such as an antimicrobial or an antineoplastic in treating a disease or condition. The term is used in the context of resistance that pathogens or cancers have ‘acquired’; that is, resistance has evolved. Heterologous expression: the expression of a gene, part of a gene, or a cluster of genes in a host organism that does not naturally have this gene or gene cluster. Recombinant DNA technology is used to introduce exogenous genes into heterologous hosts. Homologous recombination (HR): a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks (DSBs). It is also an important way of producing gene diversity. Homology arms: the short stretches of nucleotides included in linear plasmid backbones via PCR, which share homology with the target cloning sites. Homology arms define the region that is to be cloned into plasmid vectors. Natural products (NPs): compounds found in nature in general, but often used more narrowly to refer to functional secondary metabolites found in microorganisms and plants. Next-generation sequencing (NGS): also known as massively parallel or deep sequencing; a DNA sequencing technology that has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day.

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phenomenon can be explained by a number of reasons. In native hosts, most of these BGCs are under tight transcriptional regulation, resulting in poor or no expression of these BGCs under normal laboratory culture conditions. Additionally, some native hosts are difficult to culture in routine laboratory conditions and some lack genetic manipulation systems to investigate their BGCs. BGCs for NP biosynthesis vary in size from a few kilobases to more than 100 kb, even reaching up to 200 kb [12]. Therefore, it has become a challenge to utilize this vast information, to express these cryptic BGCs in compatible heterologous hosts for their functional expression, and to produce secondary metabolites from these BGCs. To functionally express these cryptic or silent BGCs, various strategies have been developed and are thoroughly reviewed elsewhere [6,13]. Here, we review Recombineering technology. This is an in vivo HR-based genetic engineering method primarily used in Escherichia coli (E. coli) by using short homology arms. In recent decades, it has been developed into a powerful and inclusive tool for the manipulation of NP BGCs. We review important advances in Recombineering, including direct cloning from complex host genomes, editing of BGCs, and retrofitting of these cryptic BGCs for functional expression.

General Genetic Engineering Based on HR Traditional molecular cloning methods mostly rely on restriction endonuclease cleavage and ligation of DNA molecules and require favorable restriction sites, which reduces the practical applications of these methods. DNA fragments need to be specifically joined by ligation either between or near favorable restriction sites. The frequent existence of restriction sites limits the length of DNA that can be engineered [14]. To overcome such disadvantages, many alternative methods for genetic engineering have been established. Among them, genetic engineering mediated by HR has emerged as a significant addition to existing technologies and has become a precise, swift, and steady way of exchanging genetic segments between two DNA molecules. The HR system of Saccharomyces cerevisiae provided an ideal platform to develop novel DNA junctions. Genetic engineering such as the commonly used transformation-associated recombination (TAR) system has been used for major breakthroughs in recombinogenic engineering [15,16]. However, its many limitations include the relatively difficult isolation of the intact yeast artificial chromosome (YAC), genetic instability of YACs, and the high degree of chimerism and low yield of purified DNA products [17]. Generally, the cloned and engineered BGC needs to be transformed into a heterologous host. From a practical perspective, isolating YACs with high purity is time consuming, which sometimes makes it difficult to obtain enough goodquality YACs to conjugate into heterologous hosts. Further engineering of recombinant DNA molecules is considered laborious using yeast compared with E. coli [18]. The endogenous HR system in E. coli is mainly mediated by the chromosome-encoded recombinases RecA/RecBCD. RecA plays an essential role in HR via the Holliday mechanism. Many strategies based on HR have been adopted to generate recombinant DNA fragments in E. coli [19,20]. RecBCD is a group of large multifunctional enzymes including the RecB, RecC, and RecD subunits. This RecBCD complex is also characterized by strong exonuclease activity, which enables RecBCD to rapidly degrade exogenous linear DNA molecules. Therefore, RecAdependent recombinogenic engineering is well suited to HR between exogenous circular DNA and the chromosome or between circular exogenous DNA molecules [21]. Linear DNA molecules can be introduced and maintain stability only in RecBCD-deficient E.coli strains, which display poor cell growth and low recombination efficiency [22,23]. RecA-dependent recombinogenic engineering requires much a longer homologous region (N500 bp).

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Recombineering: Phage-Recombinase-Mediated HR in E. coli To create a flexible and reliable HR system in E. coli, in 1998 Zhang and colleagues discovered an alternative strategy initially called ET recombination or ET cloning, later described as Recombineering ('Red/ET Recombineering' in some references). Recombineering is an in vivo method of genetic engineering primarily used in E. coli independent of RecA. Rather, it depends on pairs of phage-derived proteins, either RecE/RecT from Rac prophage or Redα/Redβ from λ phage [14]. The RecE/RecT and Redα/Redβ protein pairs are functionally similar [14,24]. RecE and Redα are 5′→3′ ATP-independent exonucleases (ExoVIII exonucleases), while RecT and Redβ are DNA annealing proteins [25]. A functional interaction between RecE and RecT or Redα and Redβ protein pairs is required for the HR reaction [26]. Another protein, Redγ, identified only in λ phage (not in Rac prophage), was found to significantly promote the recombination efficiency of Redα/Redβ and was later identified as an inhibitor of the RecB subunit of the RecBCD complex. This protein guards linear DNA molecules from degradation by endogenous nucleases [27,28]. The deduced mechanism of Recombineering technology is explained in Box 1 and Figure 2. Biological Significance of HR Based Recombineering In contrast to HR-based genetic engineering of S. cerevisiae and recA-dependent genetic engineering of E. coli, Recombineering has many advantages. First, it is quick and reliable, and it easily handles linear DNA molecules. Linear DNA molecules, in the form of either double-stranded DNA (dsDNA), which can be synthesized by PCR, or single-stranded DNA (ssDNA) in the form of synthetic oligonucleotides, can be used to provide homologous substrates [18,29]. Second, Recombineering requires comparatively shorter homologous sequences (~50 bp), which are short enough to be included in synthetic oligonucleotides. Third, compared with RecA-dependent recombinogenic engineering, Recombineering has much a higher efficiency, fewer off-target effects, and shorter time requirements (3 days for

Box 1. The Mechanism of Red/ET Recombineering RecE from prophage and Redα from λ phage degrade linear DNA molecules from the 5′ end, resulting in either a partial duplex molecule with a 3′ exposed overhang of ssDNA or leaving ssDNA if the dsDNA is shorter in length [77,78]. These resultant ssDNAs act as substrates for the ssDNA-binding proteins Redβ or RecT [79,80]. To prevent nascent ssDNA regions from further degradation, RecT/Redβ binds to ssDNA, and this DNA–protein complex is escorted to the genomic site for recombineering [79,81,82]. These recombineering sites may be supercoiled areas of the transcriptome or areas where DNA repair of damaged DNA is performed or are inside the replication fork (see Figure 2 in main text). The main differences between LLHR and LCHR lie in their deduced mechanisms: LCHR prefers ‘strand invasion’, which requires the cell to replicate to allow recombination to occur, while LLHR prefers ‘strand annealing’, which is independent of cellular replication. The mechanism of LLHR is still being investigated. Data from other experiments suggest that integration of DNA cassettes to the lagging strand in the replication fork is more efficient than integration to the leading strand. when dsDNA (usually in the form of PCR product) is used as a substrate, Redα start digesting only from one end, results into single stranded intermediate which is to be used to establish single stranded heteroduplixes at the replication fork. Based on the fact that Redα exonuclease activity requires 5' phosphorylated end, the dsDNA substrate might be designed asymmetrically so the nascent ssDNA is complementary to lagging strand. Thus, exogenous linear DNA molecules work as Okazaki fragments during the process of replication [83,84]. Through PCR, a plasmid backbone containing an origin of replication along with a selectable gene that is normally incorporated to select positively recombined clones, flanked with a homology arm to target a DNA region, can be amplified and used to target a specific region of DNA from a mixed source. DNA regions that are subcloned using this strategy and then amplified in vivo in Escherichia coli cells by the host cellular replication machinery are made more accurately, with a smaller chance of errors than during PCR-based amplification of long DNA regions. As complementary homologous sequences present on exogenous linear DNA amplified through PCR determine the site of recombineering, virtually any site on DNA in the E. coli chromosome, BACs, plasmids, or mixed sources can be genetically cloned and engineered without requiring specific restriction sites. This characteristic of recombineering omits the need for traditional DNA cloning and engineering methods.

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RecE or Redα 5′→ 3′ exonuclease

Redα 5′→ 3′ exonuclease

RecT or Redβ ssDNA binding protein

HA1

Strand invasion

HA2

Redβ ssDNA binding protein

HA1

HA2

Strand annealing

(A)

(B)

Beta recombination

(C) Trends in Biotechnology

Figure 2. Models for Red/ET mediated Recombineering. For clarity, only one linear end is shown. As both RecE and Redα are 5′→3′ double-stranded DNA (dsDNA)-dependent exonucleases, RecE or Redα degrades linear DNA molecules from the 5′ end, resulting in a dsDNA molecule with single-stranded DNA (ssDNA) 3′ exposed overhangs. These resultant single-stranded overhang DNA ends act as substrates for the ssDNA-binding protein Redβ or RecT. To prevent nascent ssDNA regions from further degradation, RecT/Redβ binds to ssDNA, and this DNA–protein complex is escorted to the specific site for recombineering. Strand invasion is the deduced mechanism for linear–circular homologous recombination (LCHR) by Redα/Redβ (A), while strand annealing is the deduced mechanism for linear–linear homologous recombination (LLHR) by RecE/RecT (B) and beta recombination is for homologous recombination between ssDNA (as Okazaki fragment) and dsDNA during the process of replication) [83,84].

Red/ET versus 2 weeks for TAR) [14,18,26]. Fourth, many precise genetic modifications, including variably sized deletion and insertion, point mutation, or alteration of DNA sequences, can be precisely performed by Recombineering without depending on the availability of restriction sites [18,30–32]. These advantages make it a simple and easy method for direct cloning and subcloning of long DNA regions without requiring restriction sites and avoids the tedious work of making and screening genomic libraries.

Advances in Recombineering for NP Biosynthesis Initially, Recombineering was developed to engineer bacterial artificial chromosomes (BACs). However, its capacity to clone large DNA fragments in E. coli worked so well that it was adapted as an ideal method for the functional characterization of long BGCs of microbial NPs. Later advances further enhanced its cloning competence and the engineering of large BGCs. Discovery of Full-Length RecE for Bioprospecting of NPs In 2012, Fu and colleagues discovered that full-length RecE and RecT protein pairs are more proficient at HR between two linear DNA molecules than at HR between linear and circular replicating DNA molecules. With the discovery of full-length RecE, this study recombined two linear DNA molecules (a linearized target DNA fragment and a PCR-amplified linear dsDNA molecule with a vector backbone flanked with homology arms to the target DNA). For the sake of convenience, they called this method linear–linear HR (LLHR) and the previous method linear–circular HR (LCHR).

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To check the practical utility of the full-length RecE/RecT protein pair, they directly cloned ten large nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) BGCs of 10– 52 kb from the genomic DNA of Photorhabdus luminescens TT01 into E. coli using expression vectors, omitting the tedious work of traditional genomic DNA library construction and screening methods, which yielded a series of microbial NPs [33]. This study also elaborates on the detailed differences between the mechanisms of LLHR and LCHR. Using full-length RecE along with RecT considerably increases the efficiency of recombineering relative to that of classical HR-based methods either by Redα/Redβ or by truncated RecE (with a deletion of 602 amino acid residues in the upstream part) and RecT. These amendments to existing Recombineering provide phenomenal opportunities to utilize the rapidly growing genome sequencing data available in public databases. Improved Seamless Mutagenesis Using cm-CcdB Counterselection and Recombineering In 2014, Wang and colleagues introduced a simple and efficient method for improved seamless mutagenesis using a two-step selection/counterselection strategy. The first step involves the insertion of a selectable cassette (normally an antibiotic-resistance gene) along with a counterselectable gene ccdB, encoding a bacterial toxin CcdB at the target genomic site. The site of insertion for the selectable cassette depends on the flanking homology sequences. Therefore, it could be virtually any site on the chromosome, BAC, or multicopy plasmid. The second step is replacing the selectable cassette (the antibiotic-resistance gene and ccdB) with the desired sequence by selecting it against the counter-selectable gene. The advantage of this system is the combination of the naturally available bacterial toxin–antitoxin (CcdB–CcdA) system with Recombineering. The first step of this process needs to be performed either in a CcdB-resistant strain of E. coli GBred-gyrA462 (which has a gyrA462Arg→Cys point mutation) or with CcdA (the antidote to the bacterial toxin CcdB)-inducible coexpression. The second step requires counterselection in CcdB-sensitive strains (GBred05 having normal gyrA or no expression of ccdA) (Figure 3). To improve the utility of this improved, seamless mutagenesis method for NP BGC characterization, earlier work introduced a point mutation in a NRPS BGC (encoding enzymes for the biosynthesis of luminmides) in a plasmid [34]. Exonuclease Combined with Red/ET Recombination (ExoCET) System Derived from Recombineering To further improve Recombineering and establish an easily handled genetic engineering platform, ExoCET was introduced for the direct cloning and bioprospecting of long DNA fragments with absolute rapidity and nucleotide precision. In ExoCET, during an in vitro reaction, T4 polymerase, which has exonuclease activity, promotes end-to-end annealing between a PCR-amplified linear cloning vector and a target DNA fragment that is released from genomic DNA. Consequently, the co-transformation efficiency is increased in E. coli. Afterwards, RecE/RecT promotes LLHR in vivo and results in precise direct cloning of the target DNA fragment. This approach is based on the idea that the efficiency of the direct cloning process can be enhanced by annealing the PCR-amplified linear vector and target genomic fragment together in vitro prior to transformation into E. coli for in vivo HR by Red/ET. This concerted action of T4pol and Red/ET is believed to be more proficient for the direct cloning of long DNA regions than either process alone. ExoCET is generally applicable to a broader range of direct clonings with respect to size (up to 106 kb so far) and genome complexity (to at least 3 × 109 bp) [35]. 6

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Figure 3. Simple Strategy for CcdB Counterselection and Red/ET-Mediated Seamless Point Mutation. Gene disruption, knockout, deletion, or point mutation can be introduced in multicopy plasmids, BACs, or chromosomes using CcdB counterselection. First, the target DNA and the linear ccdB-selection marker (ccdB-sm) cassette are co-electroporated for recombination into the CcdB-resistant strain (Escherichia coli GBred-gyrA462 or a strain with ccdA gene expression), conferring resistance to the CcdB toxin. In a second round of recombineering, the ccdB-selection marker cassette can be replaced either by single-stranded DNA (ssDNA) in the form of synthetic oligos or double-stranded DNA (dsDNA) amplified by PCR in a CcdB-sensitive strain (GB05-red or a strain without ccdA gene expression), resulting in seamless mutagenesis of target sites. The Red/ET system encoded by the genome or plasmids works in both steps.

A Modular System for Direct Cloning and Horizontal Transfer of BGC Mediated by Recombineering Since heterologous expression of NP BGCs is necessary for the functional expression of these cloned BGCs, an appropriate heterologous host needs to be determined for a particular NP BGC, and different heterologous hosts demand different horizontal gene-transfer methods. To make this technology adoptable and easily handled, a modular system was developed for direct cloning and horizontal gene transfer via transposition, conjugation, site-specific recombination

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(SSR), or retrofitting based on the cloning vector pBR322, p15A, or pBAC. The differences in the insertion capacities and copy numbers of these vectors allow BGCs of different sizes to be cloned onto them. Linear cloning vectors with homologous arms were prepared by PCR amplification using these vectors as templates for direct cloning of BGCs via LLHR by Red/ET. Then, some standardized linear DNA transfer cassettes/modules for transposition, conjugation, or site-specific integration were released from R6K-based plasmids and added into these BGC-harboring plasmids via LCHR by Red/ET, according to the particular host. This approach offers a modular and practicable platform for direct cloning and transfer of BGCs into heterologous hosts (Figure 4) [36].

Application of Recombineering in the Discovery of NP The discovery of RecE/RecT and Redα/Redβ (Red/ET in short) recombineering was an important advance in exploiting untapped treasure troves of uncharacterized BGCs for their functional characterization and for the discovery of novel microbial NPs. Besides direct cloning, Red/ET mediated Recombineering facilitates the assembly and engineering of BGCs.

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Figure 4. Operation of ExoCET, a New Version of Recombineering, for Direct Cloning and Genetic Engineering of Natural Product (NP) Biosynthetic Gene Clusters (BGCs) for Heterologous Expression. The target BGC is released from the genomic DNA with a chosen restriction enzyme or with Cas9 digestion [35]. In the first step, linear vectors containing ori and a selection marker (sm) flanked by homology arms, as illustrated (colored boxes), are used for direct cloning by linear–linear homologous recombination (LLHR) (left). The transgenic clones can then be modified by linear–circular homologous recombination (LCHR) (right) to introduce options for promoters or horizontal gene transfer cassettes, as illustrated.

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Direct Cloning and Assembly of Complete NP BGCs In 2005, Wenzel and colleagues first used Red/ET technology to rebuild an entire 43-kb PKS/ NRPS hybrid BGC from a genomic cosmid library, which resulted in the production of the NP myxochromide [37]. This technology has since been applied to the construction and heterologous expression of complete microbial NP BGCs. With the availability of more microbial genome sequences, a variety of complete NP BGCs ranging from 11 kb to 106 kb have been cloned directly from genomic DNA by Red/ET. Functional expression of these BGCs in appropriate hosts produced structurally diverse NPs (Table 1) [33,38–52]. These compounds display high diversity in their structure and bioactivity. Refactoring of BGCs for NP Biosynthesis After the molecular cloning of any BGCs, the next step in NP research is refactoring or genetic engineering to activate silent or cryptic BGCs in their heterologous hosts, and even triggering the production of unique and novel NP derivatives through combinatorial biosynthesis. Through this Red/ET technology, genes from similar clorobiocin BGCs have been replaced to produce hybrid aminocoumarin antibiotics [53]. Similarly, the novobiocin and clorobiocin BGCs have been expressed in heterologous hosts through Red/ET recombineering [54]. In another approach, a novel geldanamycin analog was produced through Red/ET recombineering and gene complementation [55]. Using Red/ET recombination, NP BGCs have been reconstituted [56], and conjugation, integration, and transposon cassettes were inserted to facilitate their heterologous expression [39,41].

Table 1. Microbial Cryptic Biosynthetic Clusters Cloned and Assembled by Recombineering NP

Native host

Size of BGC (kb)

Heterologous expression host

Refs

Chuangxinmycin

Actinoplanes tsinanensis

11.0

Streptomyces coelicolor A3(2)

[52]

Sevadicin

Paenibacillus larvae

11.6

Escherichia coli

[48]

Luminmide

Photorhabdus luminescens TT01

15.6

E. coli GB05-MtaA

[33]

Luminmycin

Photorhabdus luminescens TT01

18.3

E. coli Nissle 1917

[33]

Syringolin

Pseudomonas syringae

19.0

E. coli GB05-MtaA

[43]

Glidobactins/Luminmycins

Burkholderia DSM7029

27.8

E. coli Nissle 1917

[47]

Coumermycin A1

Streptomyces rishiriensis DSM40489

36.8

S. coelicolor M512

[42]

Bacillomycin

Bacillus amyloliquefaciens FZB42

37.2

Bacillus subtilis

[51]

Tubulysin

Cystobacter sp. SBCb004

40.0

Myxococcus xanthus

[39]

Phenalinolactone

Streptomyces sp. Tü6071

42.0

Streptomyces

[38]

Myxochromide S

Stigmatella aurantiaca

43.0

Pseudomonas putida M. xanthus

[37]

Colibactin

E. coli Nissle 1917

50.0

E. coli GB2005 E. coli GB05-MtaA

[46]

Myxothiazol

S. aurantiaca DW4-3/1

57.0

M. xanthus

[41]

Epothilone

Sorangium cellulosum

58.0

Burkholderia DSM7029

[73]

Disorazol A

S. cellulosum

58.0

M. xanthus

[50]

Salinomycin

Streptomyces albus DSM41398

106.0

S. coelicolor

[35,49]

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Red/ET technology was further used to generate a novel unglycosylated phenalinolactone derivative through genetic engineering of a pla BGC cloned from a cosmid library. Deletion of genes from the pla BGC resulted in the generation of potent derivatives [38] and unnecessary genes not required for dawenol biosynthesis were also deleted [57]. In another example, the dptBC subunit of the deptomycin BGC was altered by single/multiple module exchange and the inactivation of several tailoring enzymes resulted in the production of several novel bioactive lipopeptides [58]. Optimizing Biosynthetic Pathways and Improving the Production Titer of NPs Functional expression of cryptic or putative complete BGCs in their native hosts, or even sometimes in heterologous hosts, remains difficult for several reasons. For example, some precursor biosynthetic genes are missing, or promoters are dysfunctional in hosts for the biosynthesis of a particular NP. Therefore, their biosynthesis patterns first need to be optimized. Red/ET based recombineering has been used to overcome these adversities. Gross and colleagues reported the use of this technology to optimize the biosynthesis pattern for NPs. Pseudomonas putida is a heterologous host for many myxobacterial NPs. However, because a critical precursor, methylmalonyl coenzyme-A (mm-CoA), is not synthesized metabolically in P. putida, metabolic engineering is required for the biosynthesis of mm-CoA-dependent NPs. A putative mm-CoA biosynthetic pathway from Sorangium cellulosum So ce56 was cloned and expressed in P. putida via Red/ET technology. Furthermore, a myxothiazol BGC was cloned from two cosmids, an inducible Pm promoter was inserted upstream to the BGC, and other genetic modules required for conjugation and integration into P. putida were added via Red/ET recombineering. After integration into P. putida, myxothiazol requiring mm-CoA as an extender unit was produced [59]. Using Red/ET recombineering, a myxobacterially derived phosphopantetheinyl transferase (PPTase) gene required for biosynthesis of glidobactin was introduced into E.coli Nissle 1917, leading to the improvement of glidobactin production by tenfold [47]. After syringolin BGC was cloned from the genomic DNA of Pseudomonas syringae and a genetic organization similar to that of the native host of syringolin was constructed by the insertion of additional sylA and sylB genes via Red/ET technology, the original promoters were also replaced by a synthetic bidirectional promoter to generate syringolin analogs in E. coli [43]. Another example of optimizing the biosynthesis of a NP was demonstrated by Chai and colleagues. The native start codon of the tubC module of tubulysin BGC has a rare TTG start codon, which could limit the expression of tubulysin in heterologous hosts. In this work, TTG was replaced by ATG and engineered constructs were expressed in heterologous hosts, yielding novel tubulysin bioactive derivatives [39]. Furthermore, in 2018, Song and colleagues reconstructed an insecticide spinosad BGC by rational design of DNA sequences, DNA chemical synthesis, and multiple-fragment assembly by Red/ET recombineering, which resulted in the production titer of spinosad increasing 328-fold [60]. As mentioned previously, many microbial secondary metabolites are poorly expressed in their native hosts, so the production titer of these valuable NPs is negligible. Although heterologous expression of putative BGCs may yield many valuable NPs, improving the production of potent NPs remains a timely goal. Beyond direct cloning and capture of NP BGCs, Red/ET based recombineering has far greater potential for genetic engineering of 10

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BGCs and has been widely used to improve the yield of NPs. Via Recombineering, many groups have engineered NP BGCs by substituting native promoters with synthetic, inducible, or constitutive promoters to upregulate the transcriptional process and consequently either functionally express biosynthetic genes or yield high-titer production of potent microbial NPs [40,41,43,50,55,58,61–65].

Concluding Remarks and Future Perspectives Many molecular biology techniques have been developed recently for the functional expression of silent or cryptic BGCs. However, these techniques have had varying degrees of success [66]. Although approaches for in vivo activation of cryptic BGCs in native hosts can effectively avoid precursor availability problems, which are obstacles to the biosynthesis of a particular NP in heterologous hosts, this strategy is limited to culturable microbes and cannot be applicable to unculturable microbes or metagenome origin. Contrary to in vivo activation of silent or cryptic BGCs in native hosts, genetic manipulation can be easily achieved by direct cloning and heterologous expression of putative BGCs in non-native hosts. Emerging synthetic biology techniques have enabled researchers to directly clone large DNA assemblies while bypassing the traditional but laborious library construction and screening (Box 2). In addition to Recombineering, some of these techniques include yeastbased TAR [67] and Cas9-assisted targeting of chromosome segments (CATCH). CATCH has been applied only to prokaryotic genomes and mostly relies on primary PCR based screening; it is more laborious than Red/ET recombineering [35]. In vitro Cas9 cleavage and Gibson assembly [68] have been used to clone large DNA assemblies on BACs. Recent improvements in TAR include its combination with Cas9-assisted cleavage, but this approach has demonstrated few practical examples.

Outstanding Questions Can Red/ET recombineering work in eukaryotic cells? How can Red/ET recombineering be used to facilitate NP discovery from medicinal plants? Is it possible for a multienzyme PKS/ NRPS system to be engineered by Red/ET in vitro? Can we design a modular NP biosynthetic pathway that can produce a target specific natural product? With the increasing number of genome sequencing projects, is it possible to determine a relationship between the number of uncharacterized or cryptic NPs BGCs available in public databases and the number of NPs reported as potential drugs? How can we accelerate the genetic modification of a NP-producing strain to increase the production titer of a particular NP?

Recombineering is simple, easily handled, less time consuming (3 days for Red/ET compared with 2 weeks for TAR), and broadly applicable across a wide range of fragment sizes (up to 106 kb so far) and genome complexities (to at least 3 × 10 9 bp), which were considered limiting factors during direct cloning of large BGCs of putative NPs and their functional expression until the development of Recombineering. Recent advances in Recombineering (direct cloning, seamless mutagenesis, and assembly of DNA) can

Box 2. Working Compatibility of Recombineering with Other Technologies Since the discovery of Red/ET (RecET and Redαβ) systems, some recombineering-based genomic engineering methods have been developed, proving that Recombineering was quite efficient in many different genetic engineering studies. In 2009, Wang and colleagues developed multiplex automated genome engineering (MAGE) for large-scale cell programing based on ssDNA-binding proteins of recombineering for allelic replacement by using ssDNA or a pool of oligonucleotides [85]. MAGE was also used to develop a hierarchical conjugative assembly genome engineering (CAGE) method to replace a genomic stop codon in Escherichia coli, resulting in altered expression of targeting genes [86]. Recombineering coupled with a CRISPR/Cas9 system with dual-RNA:Cas9 targeting has been used for genome engineering in E. coli and the combination of these two methods increased the mutation efficiency [87]. Recombineering was coupled with CRISPR/Cas9 to target different loci and generate scar-less and marker-less gene replacement mutants [88]. Phage-recombineering-based MAGE coupled with CRISPR/Cas9 has also been used for metabolic engineering in E. coli [89]. Bakar and colleagues developed a gene targeting method for protein tagging called RAC (recombineering and Cas9). RAC was used for monoallelic and biallelic targeting and for protein tagging with reasonably good efficiency [90]. Finally, another study reported that SceI-created DSBs can improve the engineering efficiency when coupled with Recombineering [91,92]. These reports reflect the flexibility and utility of Red/ET recombineering with other synthetic biology techniques for characterizing and bioprospecting genomes with higher efficiency and accuracy.

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characterize BGCs rapidly, within days, while keeping pace with the rate of BGC identification. We anticipate a new and novel NP discovery paradigm in which a BGC characterization algorithm is coupled with high-throughput pathway cloning and genetic engineering methods. This paradigm will lead to the discovery of various novel NPs with clinical importance. Despite the currently reported success of Recombineering, future research (see Outstanding Questions) should be directed towards improving the cloning efficiency of BGCs larger than 100 kb. Direct cloning (rather than stitching) of the complete BGC (106 kb) for the polyether salinomycin was not successful until Recombineering was developed into ExoCET [35]. However, it is still difficult to clone directly larger NP BGCs (107–200 kb) [69] in one step using Recombineering. Similarly, further improvements are needed to clone polyketide BGCs with repetitive sequences, which sometimes lead to off-target effects using Red/ET. Along with these, research needs to be done to improve the efficiency of Recombineering for multifragment assembly of different DNA fragments, which can currently be used to assemble up to 13 fragments [60]. Recently, Wang and colleagues identified a similar recombineering system in Burkholderiales species [70], which facilitated genome mining of cryptic NP BGCs and set an example for future research to explore Red/ET-like recombination systems in other NP-producing bacteria. The identification of Red/ET-like recombination systems in other microbes will also be helpful in enhancing the NP-producing potential in different microbial species. So far, the Red/ET system can be applied to genetic manipulations in E. coli and many other prokaryotic cells [70–74] but it still cannot be used in eukaryotic cells. Medicinal plants also produce a variety of potent NPs. Hence, the Red/ET recombination system also needs to be evaluated for use in bioprospecting NP BGCs from medicinal plant genomes. Future work should also emphasize improvement of the cloning efficiency of NP BGCs from complex mixtures of DNA sources, such as metagenomic samples. However, although metagenomes contain potential cryptic BGCs from uncultivable microbes and are rich sources of new bioactive compounds [75], the low concentration of DNA makes it challenging to clone intact BGCs from such mixtures. The utility of Red/ET to mimic metagenomic samples by a diluting target genome using other bacterial genomes has been tested with some success, but there has still not been an example of a directly cloned NP BGC with Red/ET from complex environmental DNA samples, unless by the use of metagenomic DNA library screening and subsequent heterologous expression [76]. We expect that more improvements in the existing Red/ET system will bring it to the forefront of NP research. More potentially, with the increasing number of uncharacterized BGCs and decreasing cost for gene chemical synthesis, we predict that it will become easier to obtain complete NP BGCs for heterologous expression based on rational design, DNA chemical synthesis, and assembly by Red/ET, Gibson, or TAR technologies. Acknowledgments This study was supported by the National Key R&D Program of China (No. 2018YFA0900400 and 2019YFA0905700), the National Natural Science Foundation of China (31670097 and 31700073), and the Shandong Province Natural Science Foundation (ZR2017MC031, ZR2018ZC226, ZR2019JQ11, ZR2017ZB0212). We are thankful to the Open Project Program of State Key Laboratory of Bio-based Material and Green Papermaking (KF201825) and the 111 Project (B16030) for financial support. We are also obliged to Aqib Sayyed from Shandong University for the editorial assistance with the figures. We would also like to thank Liyaqat Khan from the University of Sheffield, UK for his English proofreading.

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