Combinatorial Evolution of Enzymes and Synthetic Pathways Using

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Combinatorial Evolution of Enzymes and Synthetic Pathways Using One-Step PCR Peng Jin, Zhen Kang, Junli Zhang, Linpei Zhang, Guocheng Du, and Jian Chen ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00240 • Publication Date (Web): 11 Jan 2016 Downloaded from http://pubs.acs.org on January 16, 2016

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ACS Synthetic Biology

Figure 1. Schematic diagram of the RECODE method with two-step (a) or one-step (b) PCR. Phosphorylated mutagenic oligonucleotides, UAP and DAP were annealed to template DNA after denaturation. All gaps between the primers were filled and ligated with thermostable DNA polymerase and ligation. For two-step PCR, single-stranded mutant products of the 1st step PCR were purified, and then used as the template to synthesize double-stranded variants in the 2nd step PCR reaction with AP primer which specifically matches DAP. For one-step PCR, the AP primer was added in the reaction to selectively synthesize the complementary chains of the mutant strand. The concentration of AP was set with 0.2 µM which was 2 times of that of the DAP primer. 352x139mm (300 x 300 DPI)

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Figure 2. Optimization and application of RECODE system. (a) Termination codons were introduced into galactosidase (lacZ) and esterase (estC23) with substitution or deletion of the native codons by RECODE. (b) The phenotype of the recombinants wild-type galactosidase (blue colonies) and esterase (clear halos). LB agars containing tributyrin (esterase substrate) and x-gal (galactosidase substrate) were used. (c) and (d) showed the phenotypes of variants by RECODE with two-step and one-step PCR systems, respectively. (e) Combinatorial engineering of SIR fragment of the rpoS gene. Four mutant spacers between -35 and -10 boxes of SIR segments were inserted upstream of the gfp gene to control the expression of GFP. (f) The transcriptional strength of the SIR variants with one-round evolution by RECODE (with GFP protein as a reporter). (g) Alignment of the nucleic acid sequences of the SIR variants. 160x175mm (300 x 300 DPI)


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Figure 3. Combinatorial evolution of the leech hyaluronidase by RECODE. (a) Illustration of the evolution and screening process. The activities of the wild-type LHAase and its variants towards heparin (top) and hyaluronan (bottom) were determined in 96-well microtiter plates. (b) and (c) are the enzyme activities of the variants (culture supernatants) towards hyaluronan and the non-natural substrate heparin, respectively. (d) Diverse distribution of the mutant amino acids (red) in the constructed variants. All error bars indicate ± SD, n = 3. 203x182mm (300 x 300 DPI)



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Figure 4. Combinatorial engineering the regulatory elements and pathway enzymes to optimize synthetic pathway towards ALA. (a) The heme biosynthesis pathway via ALA in E. coli. The arrows in red represent a positive relationship between ALA accumulation and overexpression of the enzymes HemA, HemL and HemF. (b) Illustration of combinatorial engineering of the promoters, ribosome binding sites and pathway genes. (c) Evaluation of the variants in 96-well microtiter plates with the parent E. coli LAF strain as control (red arrow). (d) ALA accumulation of the variants in shake flasks. All error bars indicate ± SD, n = 3. 264x188mm (300 x 300 DPI)


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Figure 5. RECODE generalized workflow for rapid construction of novel regulatory elements and enzymes, and engineering of optimized synthetic pathways to facilitate the development of in vivo and in vitro synthetic biology. 306x206mm (300 x 300 DPI)



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1

Combinatorial Evolution of Enzymes and Synthetic Pathways Using

2

One-Step PCR

3

Peng Jin†,‡, Zhen Kang†,‡,§,*, Junli Zhang†,‡, Linpei Zhang†,‡, Guocheng Du†,‡,§ and

4

Jian Chen†,‡

5

†

6

Biotechnology, Jiangnan University, Wuxi 214122, China

7

‡

8

Wuxi, Jiangsu 214122, China

9

§

The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of

Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University,

The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of

10

Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China

11

S Supporting Information ○

12 13

ABSTRACT: DNA engineering is the fundamental motive driving the rapid

14

development of modern biotechnology. Here, we present a versatile evolution method

15

termed “Rapidly Efficient Combinatorial Oligonucleotides for Directed Evolution”

16

(RECODE) for rapidly introducing multiple combinatorial mutations to the target

17

DNA by combined action of a thermostable high-fidelity DNA polymerase and a

18

thermostable DNA Ligase in one reaction system. By applying this method, we

19

rapidly constructed a variant library of the rpoS promoters (with activity of 8% to

20

460%), generated a novel heparinase from the highly specific leech hyaluronidase

21

(with more than 30 mutant residues) and optimized the heme biosynthetic pathway by 1


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combinatorial evolution of regulatory elements and pathway enzymes (2500±120 mg

23

L-1 with 20-fold increase). The simple RECODE method enabled researchers the

24

unparalleled ability to efficiently create diverse mutant libraries for rapid evolution

25

and optimization of enzymes and synthetic pathways.

26 27

KEYWORDS: Combinatorial mutagenesis; single-stranded DNA oligonucleotides;

28

directed evolution; pathway optimization; synthetic biology

29 30 31

Enzymes have attracted much attention and study in the fields of metabolic

32

engineering and synthetic biology because of their unique properties of accepting

33

complex substrates and high selectivity and specificity. In particular, enzymes have

34

been widely used for synthesis of fine chemicals, complex pharmaceuticals,

35

agrochemicals, bulk chemicals and biofuels.1 However, due to limited sources and

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performance of natural enzymes (for instance stability, substrate specificity, or

37

enantioselectivity), construction of novel robust enzymes with desired properties2-4 2



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has always been a subject of interest. However, it is not straightforward to artificially

39

design and synthesize new interchangeable parts, modules or systems with specific

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phenotypes owing to our limited understanding of the relationship between sequence

41

and function.5,6 Thus, many mutagenesis protocols including PCR based (such as

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DNA shuffling,7 StEP,8 RPR,9 RACHITT10 and Synthetic shuffling11) and non-PCR

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based (such as ITCHY12 and SHIPREC13) have been developed and applied.

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Especially in recent years, application DNA assembly technologies have greatly

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advanced significant progress in enzyme directed evolution.14-17

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Several single-stranded DNA (ssDNA) mutagenesis technologies have been

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developed to for in vivo genome engineering and in vitro enzyme evolution. An

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ssDNA mediated genome editing approach has been regarded as one of the most

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potential biotechnologies,18,19 but restricted host strains and time-consuming repetitive

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electroporation make it inapplicable to rapidly construct mutant libraries for in vivo

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enzyme evolution. In contrast, although mutagenic ssDNA oligonucleotides have

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been introduced for improving in vitro DNA shuffling methods17,20-22 and

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PCR-mediated site-mutagenesis approaches,23-27 the operation process was still

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comparatively complicated (for instance, the requirement of preparation of substrate

55

and digestion with DNase I) and the generation efficiency of positive mutants was

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still not satisfied.28 Thus, to enhance mutation efficiency and to simplify the

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mutagenesis process, several ssDNA mediated PCR methods have also been

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developed for multiple site-directed mutagenesis.29-31 However, the requirement of 3


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preparation of linear or circular ssDNAs as templates restricts its practical

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applications. Moreover, the employment of the room-temperature T4 DNA

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polymerase and ligase (which merely allows one-time of annealing, extension and

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ligation) resulted in rare products and blocked its potential applications in mutant

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library construction.29,30 Recently, a user-defined mutagenesis method PFunkel32 that

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based on the Kunkel mutagenesis protocols33,34 has been developed and applied for

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repeated introduction of designed site-directed mutations by recruiting the

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thermostable Pfu DNA polymerase and Taq DNA ligase, while the dependence of

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uracil-containing DNA plasmid template still confines its potential applications. Thus,

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how to fully utilize the advantages of synthetic oligonucleotides to efficiently

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introduce diversity at specified positions while controlling mutation frequency17 in

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small, multi-combinatorial and high-quality mutant libraries for high-throughput

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screening is still a key issue to be solved.1,4,5,35

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With the rapid development of synthetic biology,36 a wide range of artificial

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regulatory elements and biomolecular systems have been engineered for various

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useful tasks.5,37 In the past few decades, many sound strategies and tools including

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promoter library engineering,38 ribosome binding site engineering,39 modular protein

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scaffolds,40 tunable intergenic regions,41 dynamic sensor-regulators,42 multivariate

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modular pathway engineering,43,44 combinatorial transcriptional engineering45 and

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regulation with small RNAs46,47 and systems metabolic engineering48,49 have been

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developed for optimizing synthetic metabolic pathways. However, it remains a 4



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challenge to construct balanced synthetic pathways by primarily depending on

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regulation at transcriptional, post-transcriptional or translational levels,50-52 since these

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approaches often ignore the inherent shortcoming of pathway enzymes (for instance,

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stability, fitness, and inhibition by allosteric control with metabolites).2 In fact, tuning

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and optimization of enzymes are both important concepts for improving the overall

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synthetic pathway performance.50

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Here, inspired by the scarless DNA assembly method53 via ligase cycling reaction

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and the ssDNA-based mutagenesis studies,29,30,32,54 we developed a simple, rapid,

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efficient, and powerful in vitro evolution method termed “Rapidly Efficient

89

Combinatorial Oligonucleotides for Directed Evolution” (RECODE) (Figure 1). By

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applying this RECODE method with one- or two-step PCR, the strength of the

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regulatory element of the rpoS gene55 was increased 5-fold while the leech

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hyaluronidase LHAase56 was endowed with both activities towards hyaluronan and

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heparin. Concurrently, a novel strategy of combinatorial engineering of regulatory

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elements and enzymes for optimizing the synthetic pathway was developed based on

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this RECODE method. As a result, the heme biosynthetic pathway was optimized

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with a significant increase in the production of 5-aminolevulinic acid (ALA, 20-fold).

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■ RESULTS AND DISCUSSION

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The Basic Principle and Feature of the RECODE Method. The RECODE method

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is dependent on using thermostable DNA polymerase and ligase to achieve

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combinatorial mutations at multiple sites (Figure 1). The single-stranded 5


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oligonucleotides containing various desirable mutations were designed according to

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the target fragments. After repeated PCR-based thermal cycling, the phosphorylated

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single-stranded oligonucleotides annealed to the parent template DNA and extended

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to the adjacent oligonucleotide position by DNA polymerase. Then the adjacent 5'

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phosphate and 3' hydroxyl termini were ligated by Taq DNA ligase to form

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completely

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denaturation-annealing-extension-ligation temperature cycles, the DNA fragments

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with combinatorial mutations were generated in one-round evolution. Specifically, the

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upstream anchor primer (UAP), the downstream anchor primer (DAP) and the

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antisense primer (AP) with specific structures29 were designed and exclusively

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applied to amplify the variant DNA, which would be in favor of avoiding digestion

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with DpnI endonuclease and facilitating subsequent assembly (Figure 1).

matched

duplex

DNA

structures.

After

25

113

Optimization of the Constructed RECODE Reaction System for Multiple

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Site-directed Mutagenesis. To construct, evaluate and optimize the RECODE

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method, we first constructed an indicator system (Figure 2a) by co-overexpressing a

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galactosidase (encoded by lacZ) and an esterase (encoded by estC23)57. After

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induction with isopropyl-β-D-thiogalactopyranoside (IPTG), both activities of

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esterase (clear halos) and galactosidase (blue colonies) could be observed on

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Luria-Bertani (LB) agarose plate with tributyrin and X-gal (Figure 2b). In

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consideration of fidelity and avoiding the 5'-3'exonuclease (which may introduce

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non-specific mutations and remove the downstream oligonucleotides annealed on a 6



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template from 5' terminus),58 the Phusion® high fidelity DNA polymerase without the

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5'-3' exonuclease activity was selected for extension reactions. Simultaneously, the

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commonly used thermostable DNA ligases were comparatively investigated and the

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results showed that only the Ampligase thermostable DNA Ligase generated small

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amounts of mutant products (Supplementary Figure S1), which should be ascribed to

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its similar optimal pH (8.3) compared with the high fidelity Phusion® DNA

128

polymerase (9.3). Due to the incompatible working concentration of Mg2+ for the

129

Phusion® DNA polymerase and Ampligase thermostable DNA Ligase (2 mM and 10

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mM, respectively), the Mg2+ concentration was further investigated and optimized to

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reconcile the extension and ligation reactions. When Mg2+ was above 7 mM or below

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3 mM, only small amounts of mutant products were produced (Supplementary Figure

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S2a), suggesting high concentration of Mg2+ inhibits the extension activity of

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Phusion® DNA polymerase while low concentration of Mg2+ hinders the ligation

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activity of Ampligase thermostable DNA Ligase. Accordingly, 5 mM Mg2+ was

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utilized for extension and ligation in the optimized RECODE reaction buffer system

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(Supplementary methods). In addition, the melting temperature (Tm) of the mutagenic

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oligonucleotides, which depends on the flanking homology region of targets, showed

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a wide range (40−66 °C). Specifically, the Tm value was optimized at 50 °C for high

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recombination frequency (data not shown) by applying a consistent design of

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mutagenic oligonucleotides (Supplementary Table S1).

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Through systematic comparison and optimization of the components and reaction 7


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cycle conditions (Supplementary Figure S1-3), the RECODE method with two-step

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PCR was established (Figure 1a). On this basis, a color indicator system with

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three-locus lethal mutations (inactivation of galactosidase in one site and esterase in

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two sites) (Figure 2a) was designed and applied to visually examine the mutation

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efficiency of this method. Remarkably, it could be found that all the recombinant

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colonies on LB plates with X-gal and tributyrin substrates showed no parental

149

phenotype (blue color and transparent zone) (Figure 2c) and about 66.5% of the

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colonies

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(Supplementary Table S2). DNA sequencing results showed that 93 of the 116

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variants (blue colonies) were simultaneously introduced mutations at two sites of the

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estC23 gene while 109 of the 230 variants (white colonies) were simultaneously

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introduced mutations at three sites of the designed lacZ-estC23 operon (47.5%).

lost

both

lipolytic

and

galactosidase

activities

(white

colonies)

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To further simplify the operation process, a more simple RECODE method with

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one-step PCR in one reaction system was designed and established (Figure 1b). To

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guarantee the matching efficiency to the template, the DAP (60 nt) was designed with

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a 40 nt complementary sequence to the template and 20 nt to the AP segment (Figure

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1b). Meanwhile, a two-fold molar excess of APs over other primers were added with a

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final concentration of 0.2 µM to guarantee extension and synthesis of all the mutant

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single-strand

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(Supplementary Figure S4) gave rise to fewer recombinants, the proportion of the

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white colonies (about 61.6%, Figure 2d) was similar with that of the two-step PCR

chains.

Although

comparatively

less

8


target

PCR

products


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systems. Furthermore, DNA sequencing results showed that 26 of the 45 mutants with

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color colonies were simultaneously introduced mutations at three sites of the designed

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lacZ-estC23 operon (57.8%, Figure 2d) which was higher than that of the two-step

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PCR systems. Taken together, all the results demonstrate the high efficiency of the

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RECODE method with one-step or two-step PCR system.

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Application of RECODE to Rapidly Construct a Diverse Mutant Library of

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Regulatory Elements. Promoter engineering is a powerful strategy in metabolic

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engineering and synthetic biology.59 The transcriptional sigma factors and promoters

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have been engineered for pathway construction and optimization.60 Here, four spacer

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regions between the -35 and -10 elements from the 5′-untranslated region of rpoS

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gene (encoding the sigma factor 38 for stress resistance in Escherichia coli) was

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cloned and engineered by the RECODE method (Figure 2e) to generate a mutant

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library with different transcriptional strengths. Specifically, the green fluorescent

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protein that encoded by gfp was chosen as a reporter for visualized analysis of the

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mutant library. After one-round evolution, five hundred colonies with different visual

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green fluorescence intensities were randomly selected and cultured in 96-well plates

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for characterization. The variants that showed significant differences compared to the

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wild-type counterpart were then further confirmed in shake flask cultures. As shown

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in Figure 2f, 74 variants exhibited a broad span of fluorescence intensity (8–460% of

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the wild type). DNA sequencing results of the randomly picked variants showed that

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the introduced multiple-cassette mutations covered the four spacer regions (Figure 2g), 9


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demonstrating its high efficiency in combinatorial editing of DNA segments.

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Collectively, these data suggest that this simple RECODE method should have wide

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potential applications in rapid enzyme evolution.

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Application of RECODE to Rapidly Broaden the Substrate Specificity of the

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Leech Hyaluronidase. As an application example, a novel leech hyaluronidase

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(LHAase, without activity to heparin) secreted by a recombinant Pichia pastoris56 was

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chosen as a candidate for rapid evolution by RECODE (Figure 3a). The objective is to

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rapidly create variants with improved LHAase activities and/or broadened substrate

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specificity to heparin. According to the phylogenetic relationship and conserved

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domains among the hyaluronidases and heparanases,56 10 mutagenic oligonucleotides

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with degenerate sequences (Supplementary Table S1) were designed to cover the 10

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candidate regions near the conserved amino acids.56 After one-step PCR evolution and

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assembly, all the mutants were transformed into P. pastoris GS115. Subsequently,

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Seven hundred colonies were firstly picked in 96 deep-well plates for cultivation and

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assay of HAase activity. Then, the variants with significant differences were further

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investigated in flask cultures. Remarkably, many variants with different activities

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towards hyaluronan and heparin were successfully created after one-round evolution

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by the RECODE method (Figure 3a). In comparison, the variant M2D7 showed a

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2.4-fold increase of the LHAase activity while both the variants M4A4 and M5B4

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showed increased LHAase activity and heparanase activity (Figure 3b and 3c).

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Impressively, although the LHAase activity of the variant M4A12 decreased, the 10



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heparanase activity increased with the highest titer of 750 ± 20 U mL–1, indicating its

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potential to be an exclusive “heparanase”. Further DNA sequencing analysis of these

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variants showed that the introduced mutation sites covered all the area of candidates

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with random combinations (Figure 3d). In particular, more than 30 amino acid

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residues in a single variant were simultaneously mutated (accounting for 6.12% of the

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LHAase, 489 amino acids), demonstrating the powerful capacity of this RECODE

212

method in enzyme directed evolution.

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Combinatorial Engineering of Regulatory Elements and Enzymes to Optimize

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Synthetic Pathway. In many cases, it is not possible to optimize a synthetic pathway

215

only by engineering the regulatory elements (such as promoters and RBS). Here,

216

based on the established simple RECODE method, a novel approach of combinatorial

217

engineering of regulatory elements and pathway enzymes was proposed to optimize

218

synthetic pathways towards target compounds. As an example, the heme biosynthesis

219

pathway was simultaneously engineered at the transcriptional and protein levels for

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efficient accumulation of the intermediate ALA (Figure 4a). Specifically, the three

221

crucial genes hemA, hemL and hemF61 were simultaneously engineered at the

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transcriptional and protein levels by the RECODE method (Figure 4b). After

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one-round evolution, 1000 recombinant colonies were characterized in 96 deep-well

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plates (Figure 4c) and further investigated with flask cultures. As shown in Figure 4d,

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the recombinant BL21 (DE3) strain containing the variant A24-H6 accumulated ALA

226

to the highest titer (2500 ± 120 mg L–1) which was 20-fold of that of the parental 11


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strain. Furthermore, DNA sequencing results of eight variants (Figure 4d) showed that

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the mutation sites covered most of the designed regions including promoters, RBS

229

regions and the open reading frames of hemA, hemL and hemF (Supplementary Figure

230

S6). The results demonstrate the high efficiency of this simple approach in pathway

231

engineering. Meanwhile, the results also confirmed that fine-tuning of the three

232

committed enzymes HemA, HemL and HemF are critical for ALA accumulation.61

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Discussion. Enzyme directed evolution technologies have achieved great progress

234

in the past two decades, however, the sizes of the generated mutant libraries are

235

generally too large to fully explore by available selection strategies.50,62 Thus,

236

development of simple, fast and efficient techniques that allow rational design and

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target engineering of smaller libraries with higher degrees of precision is always a

238

major requirement.1 In the present study, a designated RECODE method with

239

one-step PCR has been developed for rapid DNA editing (Figure 1), which has been

240

demonstrated to be effectively applied in engineering of regulatory elements, enzymes

241

and synthetic pathways.

242

Different from previous ssDNA-based methods,29,30 the RECODE method was

243

developed by employing the thermostable Phusion® high fidelity DNA polymerase

244

and Taq DNA ligase (Figure 1). Combined action of the thermostable high-fidelity

245

DNA polymerase and ligase not only facilitates all kinds of DNA templates (eg.

246

double-stranded DNA fragments, cDNA, genomic DNA and plasmid DNA) but also 12



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247

avoids or reduces non-specific mutations and incorrect ligations. Here, to construct

248

this reaction system in one tube, the Mg2+ concentration and pH were demonstrated as

249

the major parameters that affect the activities of polymerase and ligase

250

(Supplementary Figure S1, 2) and optimized to achieve accordant extension and

251

ligation activities for the production of amounts of variants with desirable diversities

252

(Supplementary Figure S2). After further optimization of the extension and ligation

253

cycles (to fully anneal the mutagenic oligonucleotides and make full use of the

254

advantages of the thermostable DNA Ligase.53) and the Tm value of the homologous

255

sequence, a sufficient quantity of mutants with multi-combinatorial mutations were

256

rapidly generated for high-throughput screening. In comparison, it would be

257

impossible to achieve combinatorial multiple-cassette mutagenesis by further

258

modification of previous reported protocols because of application of the

259

room-temperature T4 DNA polymerase and ligase.29,30 Moreover, the PFunkel

260

method32 may be laborious due to the delayed addition of reaction reagents (including

261

polymerase and ligase) and degradation of the uracil-containing template and DNA

262

products not in the desired covalently closed circular by uracil DNA glycosylase and

263

exonuclease III.32

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For enzyme evolution, the size and the diversity of the created library are always

265

the critical characterizations for the subsequent screening especially when no effective

266

selection methods are available. To this end, specific upstream and downstream

267

anchor primers and an antisense primer were designed and standardized (Figure 1). 13


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Compared with previous DNA shuffling methods,7,63 the RECODE method enabled

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rapid DNA evolution with higher diversity within a smaller library size. This is

270

accomplished by the application of specific primers in the reaction system that result

271

in synthesis of the full-length active mutants without the repeat amplification of the

272

existing ones. Furthermore, this RECODE method also showed powerful capacity in

273

combinatorial editing of a target DNA with insertion and deletion of interested

274

elements (Supplementary methods and Supplementary Figure S5). Compared with

275

previous DNA shuffling strategies, this RECODE method was much more rational in

276

protein engineering especially with the assistance of crystal structure analysis. As an

277

example, we successfully created a novel “heparanase” from the highly specific leech

278

hyaluronidase (Figure 3a). Moreover, the novel approach devised for rapid

279

optimization of the synthetic pathways by combinatorial engineering of regulatory

280

elements and pathway enzymes also showed high efficiencies (Figure 4d). Compared

281

with other pathway engineering strategies that mainly focused on transcriptional

282

regulation,64-66 this approach should be comparatively easier to use in constructing an

283

optimized pathway by simultaneously improving pathway enzyme properties and

284

optimizing the concentration ratios.

285

Conclusion. We have developed a simple (one-step PCR with any form of DNA

286

templates), rapid (one-round evolution of 5 kb DNA within 3 hours), efficient (higher

287

amount of products) and powerful (multiple single-directed mutations, insertions or

288

deletions and combinatorial multiple-cassette mutagenesis) RECODE method, which 14



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289

enables us to easily reedit the target DNA to construct novel regulatory parts and

290

modules (Figure 5). Combining with protein synthesis in vitro,67,68 computer-aided

291

design69 and simulation, and efficient assembly techniques,1,70 especially the Gibson

292

isothermal assembly,71 Ligase Cycling Reaction53 and the DNA assembler,72 this

293

RECODE method will greatly promote protein studies and benefit our understanding

294

of the relationship between sequence and enzyme function. In conjunction, the

295

demonstrated approach for simultaneous modification of regulatory elements and

296

pathway enzymes will also enable us to rapidly optimize the synthetic pathways

297

towards the target products (Figure 5). Hence, the RECODE method and the derived

298

approach for pathway engineering will be of great benefit to a variety of applications

299

in the field of enzyme engineering, metabolic engineering and synthetic biology.

300

301

Bacterial Strains and Plasmids. E. coli DH5α and pBBRMCS2 (Stratagene, La Jolla,

302

CA, USA) were used for cloning and characterization of the library of SIR regulatory

303

element.55 The pMD19-T (Takara Ltd., Otsu, Japan) vector was used for cloning of

304

the lacZ-estC23 and gfp fragments. P. pastoris GS115 (his4) and the pPIC9K vector

305

(Invitrogen, Carlsbad, CA, USA) were used for expression of the hyaluronidase

306

mutant library. The pRSFduet-1 (Novagen Darmstadt, Germany) and E. coli BL21

307

(DE3) (Stratagene) were used as the vector and host, respectively, for pathway genes

308

expression of ALA production.

MATERIALS AND METHODS

15


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309

Media and Culture Conditions. LB medium was used for cloning and cell culture.

310

LB plates supplemented with 100 µg mL-1 ampicillin, 0.1 mM IPTG, 20 µg mL−1

311

X-gal (Sigma-Aldrich, St. Louis, USA), and 1% (v/v) tributyrin (Merck, Darmstadt,

312

Germany) were used for characterizing the indicator system of lacZ-estC23 at 37 °C.

313

The secondary assay of E. coli libraries was carried out in shake flask with LB broth

314

at 37 °C with 200 rpm shaking. P. pastoris was selected on MD medium (0.34% YNB,

315

20 g L−1 glucose and 15 g L−1 agar) supplemented with 2 mg mL-1 G418. Shake flask

316

cultures of recombinant P. pastoris strains were carried out at 30 °C at 200 rpm with

317

buffered methanol minimal yeast medium (BMMY, 10 g L−1 yeast extract, 20 g L−1

318

peptone, 100 mM potassium phosphate, 0.34% YNB, 10 g L−1 (NH4)2SO4, 1%

319

methanol). For production of ALA, the recombinant E. coli BL21 (DE3) cells were

320

grown at 37 °C at 200 rpm with mineral basal medium (g L−1): 20.0 glucose, 2.0 yeast

321

extract, 16.0 (NH4)2SO4, 3.0 KH2PO4, 16.0 Na2HPO4•12H2O, 1.0 MgSO4•7H2O and

322

0.01 MnSO4•H2O, pH 7.0. When necessary, 50 µg mL−1 kanamycin and 0.1 mM

323

IPTG were supplemented.

324

Primer Design. All primers (BGI; Beijing, China) in this study are listed in

325

Supplementary Table S1. The lethal mutations for galactosidase encoding gene lacZ

326

and esterase encoding gene estC23 were designed by introduction of termination

327

codons into one site of lacZ and two sites of estC23 (Figure 2a). Specifically, the

328

bases GAA, TAC, and ATGA were replaced with the termination codons TAA, TAA,

329

and TGA, respectively. To evaluate the insertion and deletion capacity of the 16



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

330

RECODE method, the oligonucleotides that contain 12 nt (ACATGCCACTGG) and

331

9 nt (TACTGGCAC) fragments for insertion at 196 and 601 sites, and a 6 nt

332

(ACACAA) fragment for deletion at 607-612 site of gfp gene were designed,

333

respectively. For editing the SIR regulatory element, four degenerate oligonucleotides

334

were designed to cover the spacer fragments between the −35 and −10 boxes of the

335

corresponding four natural tandem promoters. For combinatorial evolution of the

336

leech hyaluronidase, mutation sequences instead of the inherent triplet code were

337

introduced into 10 candidate sites near the conserved amino acids, Specifically, the

338

triplet-codon was consciously avoided the introduction of the termination codons (for

339

example VNN; V = A, C, G; N = A, T, C, G). For combinatorial evolution of the

340

ALA pathway, multiple regions of the three pathway genes (hemA, hemL and hemF)73

341

were simultaneously modified with the highly degenerate triplet-code sequence (for

342

example VNN or NNN in oligonucleotides). The ribosome binding site (RBS) regions

343

were modified with degenerate RBS sequences (DDRRRRRDDDD; D = A, G, T; R =

344

A, G), and the spacer sequences between −35 and −10 boxes flanking the hemF

345

promoter were edited with the degenerate sequence (NNNNNNNNNNNNNNNNN).

346

RECODE Manipulation. All the synthetic mutagenic oligonucleotides and the

347

downstream anchor primers (DAP) were mixed with the same concentration and

348

phosphorylated. Specifically, phosphorylation was performed in 50-µL reactions

349

containing 300 pmol of a primer mixture, 1× T4 DNA ligase buffer (NEB, New

350

England Biolabs, USA), and 8 U polynucleotide kinase (NEB). The reaction was 17


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351

incubated for 30 min at 37 °C and subsequently terminated by heating for 10 min at

352

75 °C.

353

The RECODE reaction was carried out in 50-µL system containing 0.1 µM of each

354

phosphorylated mutagenic oligonucleotide and the upstream anchor primer (UAP),

355

0.2 µM antisense primer , 0.01 pmol DNA template, 1 U Phusion DNA polymerase

356

(NEB), 5 U Ampligase thermostable DNA ligase (Epicentre, USA) and 1× optimized

357

RECODE reaction buffer (20 mM Tris-HCl, 25 mM KCl, 0.5 mM NAD, 2 mM

358

dNTPs, 5 mM Mg2+, 0.1% Triton X-100, pH 8.3). The one-step reaction system

359

(Figure 1b) was performed with the following PCR conditions: 2 min at 94 °C; 25

360

cycles of 30 s at 94 °C, 30 s at 50 °C, 2 min at 72 °C, and 3 min at 66 °C; a hold

361

period at 4 °C. In the two-step RECODE system (Figure 1a), twenty-five cycles were

362

programed in the 1st step PCR to ensure that the extended mutagenic primers were

363

fully annealed and to generate variants with different combinatorial mutations. After

364

purification, all the PCR products were applied as the templates in the 2nd step PCR.

365

Then the double-stranded products were generated with an antisense primer AP in a

366

new 50-µL PCR system containing the purified 1st step PCR products, 1 µL of

367

antisense primer and 25 µL of 2 × SuperPfu PCR MasterMix (Hangzhou Biosci Co.,

368

Ltd, China). The PCR reaction was performed with the following program: 2 min at

369

94 °C; then 3 cycles of 30 s at 94 °C, 30 s at 50 °C and 90 s at 72 °C; 5 min at 72 °C

370

and a hold period at 4 °C.

371

PCR products were run on 1% agarose gels containing EtBr at 90 V for 45 min. 18



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Page 24 of 37

372

Bands of the correct size were excised and puriﬁed with a gel extraction kit, according

373

to the manufacturer’s protocol. When applying plasmid DNA as templates, 1 µL DpnI

374

endonuclease (All endonucleases supplier, Fermentas, USA) was added in the PCR

375

products and incubated at 37 °C for 10 min to digest the template DNA.

376

Variants Library Construction and Screening. To obtain colony libraries,

377

double-stranded DNA variants produced by RECODE were cloned into vectors by the

378

enzymatic assembly DNA method.71 High-throughput screening of the library was

379

performed in 96-well microplates, and then selected variants of all the following

380

libraries were further examined in shake flasks. The lacZ-estC23 mutant library was

381

subcloned into the pMD19-T vector, and then transformed into DH5α. The library

382

was selected on LB plates supplemented with 100 µg mL-1 ampicillin, 0.1 mM IPTG,

383

20 µg mL−1 X-gal, and 1% tributyrin. The diversity of the mutant library was

384

characterized by phenotypic analysis and DNA sequencing. To construct the deletion

385

or insertion libraries of gfp gene by RECODE, the purified products were subcloned

386

into the pMD19-T vector, and then transformed into DH5α. The deletion or insertion

387

libraries were selected on LB plates supplemented with 100 µg mL-1 ampicillin,

388

respectively.

389

To construct the characterization library of the SIR variants, the gfp fragment was

390

firstly

391

EcoRI/BamHI

392

pBBR1MCS2-gfp. Then the SIR mutant library was assembled into the linear

subcloned

into

the

restriction

low-copy-number enzymes,

yielding

plasmid the

19


pBBR1MCS-2 recombinant

with

plasmid

Page 25 of 37

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393

pBBR1MCS2-gfp via enzymatic one-step assembly to control expression of the

394

reporter gene gfp. The recombinant plasmids carrying the SIR library were

395

transformed into E. coli DH5α competent cells and cultured on LB plates

396

supplemented with 50 µg mL−1 kanamycin. Screening was performed by

397

high-throughput batch-cultures in 96-well microtiter plates. Transformants were

398

picked into microtiter wells with a Qbot robotic colony picker (Genetix, Hampshire,

399

UK) and grown in LB broth at 37 °C at 200 rpm for 24 h. To analyze the activity of

400

the promoter library, cells were collected by centrifugation (1, 3000 g, 5 min) and the

401

cell pellet washed three times with sterile water. The fluorescence intensity value of

402

per OD600 cells was measured by excitation at 490 nm and emission at 530 nm using

403

an Ultra Multifunctional Microplate Reader (Tecan, Durham, NC, USA). Then, some

404

variants were further verified in shake flask culture.

405

The hyaluronidase gene H6LHyal variants were inserted into pPIC9K under the

406

control of the AOX1 promoter by enzymatic assembly. The recombinant plasmids

407

were linearized with SalI and transformed into P. pastoris GS115 competent cells by

408

electroporation. The cells were cultured on MD plates supplemented with 2 mg mL−1

409

G418. Variants were picked into microtiter wells and cultured in BMMY broth

410

containing methanol (1% v/v) at 30 °C at 200 rpm for 72 h. The cultured supernatants

411

of variants were collected and diluted for the enzymatic assay in 96-well microtiter

412

plates. The selected variants were further cultured in shake flask rocking at 200 rpm at

413

30 °C according to the previous manipulation of shake flask culture.56 The enzymatic 20



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Page 26 of 37

414

reaction containing a 1.6 mg mL−1 of the substrate (hyaluronic acid or heparin,

415

Sigma-Aldrich, USA) and an appropriate amount of enzyme in 50 mM citrate buffer

416

(pH 5.5) was incubated at 38 °C for 10 min. The reaction was terminated by heat

417

inactivation then examined using the DNS method. Colorimetric reactions were

418

measured by using the Ultra Multifunctional Microplate Reader at 540 nm. One unit

419

of hyaluronidase activity was defined as equal to the reducing power of glucuronic

420

acid (glucose equivalents in micrograms) liberated per hour from HA at 38 °C and pH

421

5.5. One unit of heparanase activity was defined as equal to the reducing power of

422

oligosaccharides (glucose equivalents in milligrams) liberated per hour from HA at

423

30 °C and pH 5.5.

424

The

three

separated

mutant

libraries

(RBS/hemL,

RBS/hemA

and

425

promoter/RBS/hemF) of the ALA pathway were assembled into pRSFduet-1 via

426

enzymatic assembly and then transformed into BL21 (DE3) competent cells. To

427

analyze the ALA yields from the variants, high-throughput screening was carried out

428

in microtiter wells with mineral basal medium broth at 37 °C at 200 rpm for 30 h. The

429

candidate variants were further verified in shake flask cultures. The production of

430

ALA was analyzed by using the modified Ehrlich’s reagent.74

431

■ ASSOCIATED CONTENT

432

S Supporting Information ○

433

Supplementary data including figures, tables and methods associated with this article

434

can be referenced in the supplementary materials. This material is available free of 21


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435

charge via the Internet at http://pubs.acs.org.

436

AUTHOR INFORMATION

437

Corresponding Author

438

Zhen Kang, *E-mail: [email protected]

439

Notes

440

The authors declare no competing ﬁnancial interest.

441

■ ACKNOWLEDGMENTS

442

This work was financially supported by the Major State Basic Research Development

443

Program of China (973 Program, 2014CB745103), the Natural Science Foundation of

444

Jiangsu Province (BK20141107), a grant from the Key Technologies R & D Program

445

of Jiangsu Province, China (BE2014607); Program for Changjiang Scholars and

446

Innovative Research Team in University (No. IRT_15R26); China Postdoctoral

447

Science Foundation funded project (125960).

448

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Figures legends

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647

Figure 1. Schematic diagram of the RECODE method with two-step (a) or one-step

648

(b) PCR. Phosphorylated mutagenic oligonucleotides, UAP and DAP were annealed

649

to template DNA after denaturation. All gaps between the primers were filled and

650

ligated with thermostable DNA polymerase and ligation. For two-step PCR,

651

single-stranded mutant products of the 1st step PCR were purified, and then used as

652

the template to synthesize double-stranded variants in the 2nd step PCR reaction with

653

AP primer which specifically matches DAP. For one-step PCR, the AP primer was

654

added in the reaction to selectively synthesize the complementary chains of the

655

mutant strand. The concentration of AP was set with 0.2 µM which was 2 times of

656

that of the DAP primer.

657

Figure 2. Optimization and application of RECODE system. (a) Termination codons

658

were introduced into galactosidase (lacZ) and esterase (estC23) with substitution or

659

deletion of the native codons by RECODE. (b) The phenotype of the recombinants

660

wild-type galactosidase (blue colonies) and esterase (clear halos). LB agars containing

661

tributyrin (esterase substrate) and x-gal (galactosidase substrate) were used. (c) and (d)

662

showed the phenotypes of variants by RECODE with two-step and one-step PCR

663

systems, respectively. (e) Combinatorial engineering of SIR fragment of the rpoS

664

gene. Four mutant spacers between -35 and -10 boxes of SIR segments were inserted

665

upstream of the gfp gene to control the expression of GFP. (f) The transcriptional

666

strength of the SIR variants with one-round evolution by RECODE (with GFP protein

667

as a reporter). (g) Alignment of the nucleic acid sequences of the SIR variants. 30



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

668

Figure 3. Combinatorial evolution of the leech hyaluronidase by RECODE. (a)

669

Illustration of the evolution and screening process. The activities of the wild-type

670

LHAase and its variants towards heparin (top) and hyaluronan (bottom) were

671

determined in 96-well microtiter plates. (b) and (c) are the enzyme activities of the

672

variants (culture supernatants) towards hyaluronan and the non-natural substrate

673

heparin, respectively. (d) Diverse distribution of the mutant amino acids (red) in the

674

constructed variants. All error bars indicate ± SD, n = 3.

675

Figure 4. Combinatorial engineering the regulatory elements and pathway enzymes to

676

optimize synthetic pathway towards ALA. (a) The heme biosynthesis pathway via

677

ALA in E. coli. The arrows in red represent a positive relationship between ALA

678

accumulation and overexpression of the enzymes HemA, HemL and HemF. (b)

679

Illustration of combinatorial engineering of the promoters, ribosome binding sites and

680

pathway genes. (c) Evaluation of the variants in 96-well microtiter plates with the

681

parent E. coli LAF strain as control (red arrow). (d) ALA accumulation of the variants

682

in shake flasks. All error bars indicate ± SD, n = 3.

683

Figure 5. RECODE generalized workflow for rapid construction of novel regulatory

684

elements and enzymes, and engineering of optimized synthetic pathways to facilitate

685

the development of in vivo and in vitro synthetic biology.

686

Supplementary data

687

Supplementary data associated with this article can be found in the online version. 31


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Combinatorial Evolution of Enzymes and Synthetic Pathways Using

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