Cas9-based genome editing system for Rhodococcus ruber TH

Journal Pre-proof A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH Youxiang Liang, Song Jiao, Miaomiao Wang, Huimin Yu, Zhongyao Shen PII:

S1096-7176(19)30266-6

DOI:

https://doi.org/10.1016/j.ymben.2019.10.003

Reference:

YMBEN 1611

To appear in:

Metabolic Engineering

Received Date: 27 June 2019 Revised Date:

10 October 2019

Accepted Date: 10 October 2019

Please cite this article as: Liang, Y., Jiao, S., Wang, M., Yu, H., Shen, Z., A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH, Metabolic Engineering (2019), doi: https:// doi.org/10.1016/j.ymben.2019.10.003. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of International Metabolic Engineering Society.

1

A CRISPR/Cas9-based genome editing system for Rhodococcus ruber TH

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Youxiang Liang1, 2, Song Jiao1, 2, Miaomiao Wang1, 2, Huimin Yu1,

3 4 5 6 7 8 9

2, 3*

Zhongyao Shen1, 2 1 Department of Chemical Engineering, Tsinghua University, Beijing 100084, China 2 Key Laboratory of Industrial Biocatalysis (Tsinghua University), Ministry of Education, Beijing 100084, China 3 Center for Synthetic and Systems Biology, Tsinghua University, Beijing 100084, China

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*Corresponding author:

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Telephone: 86-10-62795492, Fax: 86-10-62770304,

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E-mail: [email protected]

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Address: Yingshi Building 412, Tsinghua University, Beijing, China, 100084

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Manuscript prepared for submission to:

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Metabolic Engineering

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and

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Abstract

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Rhodococcus spp. are organic solvent-tolerant strains with strong adaptive

21

abilities and diverse metabolic activities, and are therefore widely utilized in

22

bioconversion, biosynthesis and bioremediation. However, due to the high

23

GC-content of the genome (~70%), together with low transformation and

24

recombination efficiency, the efficient genome editing of Rhodococcus remains

25

challenging. In this study, we report for the first time the successful establishment of a

26

CRISPR/Cas9-based genome editing system for R. ruber. With a bypass of the

27

restriction-modification system, the transformation efficiency of R. ruber was

28

enhanced by 89-fold, making it feasible to obtain enough colonies for screening of

29

mutants. By introducing a pair of bacteriophage recombinases, Che9c60 and Che9c61,

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the editing efficiency was improved from 1% to 75%. A CRISPR/Cas9-mediated

31

triple-plasmid recombineering system was developed with high efficiency of gene

32

deletion, insertion and mutation. Finally, this new genome editing method was

33

successfully applied to engineer R. ruber for the bio-production of acrylamide. By

34

deletion of a byproduct-related gene and in-situ subsititution of the natural nitrile

35

hydratase gene with a stable mutant, an engineered strain R. ruber THY was obtained

36

with reduced byproduct formation and enhanced catalytic stability. Compared with

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the use of wild-type R. ruber TH, utilization of R. ruber THY as biocatalyst increased

38

the acrylamide concentration from 405 g/L to 500 g/L, reduced the byproduct

39

concentration from 2.54 g/L to 0.5 g/L, and enhanced the number of times that cells

40

could be recycled from 1 batch to 4 batches.

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Keywords: Rhodococcus; CRISPR/Cas9; genome editing; restriction-modification

43 44

system; recombinase; acrylamide bio-production

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1. Introduction

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Rhodococccus spp. are non-sporulating, aerobic actinomycetes widely distributed

47

in environments with different nutritional conditions (Bell et al., 1998). They possess

48

excellent adaptation abilities and diverse metabolic activities, enabling them to

49

degrade and assimilate a wide range of organic pollutants, including nitriles,

50

halogenated hydrocarbons and numerous aromatic compounds (Bell et al., 1998;

51

DeLorenzo et al., 2018; Warhurst and Fewson, 1994; Xu et al., 2018). The desirable

52

traits make Rhodococcus an ideal candidate for the bioremediation of contaminated

53

environments and a wide range of biotransformations, such as enantioselective

54

synthesis and the production of amides from nitriles (Bell et al., 1998; MethCohn and

55

Wang, 1997; Nagasawa et al., 1993; Wang, 2015; Warhurst and Fewson, 1994). The

56

bio-production of acrylamide from acrylonitrile using Rhodococcus harboring nitrile

57

hydratase has been one of the most successful cases of industrial biotechnology

58

(Nagasawa et al., 1993). Rhodococcus is also an ideal chassis for the production of

59

lipids and biofuels from lignocellulosic biomass, owing to its adaptable tolerance

60

against biomass breakdown products (e.g., furans, halogenated compounds and

61

phenolics) and diverse enzymatic pathways for the assimilation of aromatic

62

compounds (DeLorenzo et al., 2018; Henson et al., 2018). Therefore, the genetic and

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metabolic engineering of Rhodococcus are of great interest, and efficient genetic tools

64

are urgently required.

65

Although various genetic toolkits such as promoter libraries, reporter genes and

66

shuttle vectors have been developed for Rhodococcus (DeLorenzo et al., 2017;

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DeLorenzo et al., 2018; Jiao et al., 2018), the genome editing of Rhodococcus remains

68

challenging, due to the high GC content (~70%) in genome, low transformation

69

efficiency and recombination efficiency. For example, the transformation efficiency of

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most Rhodococcus is usually no more than 105 cfu/µg DNA (Duran, 1998; Sekizaki et

71

al., 1998; Shao et al., 1995), thereby hindering gene knockout that involves rare

72

homologous recombination (Suzuki and Yasui, 2011). In addition, severe illegitimate

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recombination was found in Rhodococcus, and caused random integration into the

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genome when a non-replicating vector was introduced (Desomer et al., 1991).

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Recently, a recombineering method using the recombinases Che9c60 and Che9c61

76

derived from mycobacteriophage (van Kessel and Hatfull, 2007) and a linear

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double-stranded DNA (dsDNA) template flanking antibiotic resistance genes, was

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established in R. opacus PD630 with improved editing efficiency (DeLorenzo et al.,

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2018). However, the antibiotic resistance cassettes could not be recovered from the

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genome, which limited multiple rounds of genome editing. A two-step

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double-crossover recombination can recover antibiotic markers but requires an

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efficient counter-selection marker. Although various counter-selection markers,

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including upp, sacB and mazF, have been developed for different strains, most of

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them have limited applicability. For example, upp requires the host strains to be free

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of the native upp gene (Shi et al., 2013), and mazF requires a strictly inducible

86

promoter for expression (Zhang et al., 2006). SacB has been utilized in many bacteria,

87

including Escherichia coli (Crouzet et al., 1997), Corynebacterium glutamicum

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(Schafer et al., 1994), Mycobacteria (Pelicic et al., 1996), and some Rhodococcus

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(van der Geize et al., 2001). However, it did not function in some Rhodococcus such

90

as Rhodococcus equi (van der Geize et al., 2008), and the transformation of the

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suicide plasmid pK18mobSacB into R. ruber could not effectively confer a

92

sucrose-sensitive phenotype. Thus, an efficient and scarless genome editing method

93

for Rhodococcus still remains to be developed.

94

During the past several years, the clustered regularly interspaced short

95

palindromic repeat (CRISPR) system has emerged as a powerful tool for genome

96

editing in humans, animals, plants and microorganisms (Wang et al., 2016). The type

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II CRISPR/Cas9 system from Streptococcus pyogenes is the most widely used and

98

well-characterized CRISPR system (Choi and Lee, 2016; Wang et al., 2016). Cas9

99

forms a ribonucleoprotein complex with sgRNA (the fusion of crRNA and tracrRNA)

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and introduces double-strand breaks (DSBs) on the target DNA with a protospacer

101

adjacent motif (PAM) (Jinek et al., 2012). The DSBs could be repaired by

102

nonhomologous end joining (NHEJ) or homologous recombination, and consequently,

103

the target genes would be inactivated. The CRISPR/Cas9 system has been employed

104

in many microorganisms, including E. coli (Jiang et al., 2015), Saccharomyces

105

cerevisiae (DiCarlo et al., 2013), Bacillus subtilis (Altenbuchner, 2016), Streptomyces

106

(Cobb et al., 2015) and C. glutamicum (Cho et al., 2017), but has not been used in

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Rhodococcus. Recently, the CRISPR interference (CRISPRi) system was developed

108

in R. opacus PD630 for gene knockdown (DeLorenzo et al., 2018). However, to

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construct valuable industrial Rhodococcus strains requiring permanent and stable

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changes, gene knockout rather than gene knockdown is necessary. From CRISPRi to

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CRISPR, there are still great obstacles to overcome, including not only having Cas9

112

effectively work in the target strain and achieving enough transformation efficiency to

113

obtain mutants, but also achieving high recombination efficiency necessary for the

114

repair of DSBs.

115

In this study, we successfully developed a CRISPR/Cas9-based Rhodococcus

116

genome editing method with a triple-plasmid system for efficient and scarless genome

117

editing, including gene deletion, insertion and mutation. With this powerful tool, we

118

further accomplished the recombineering of R. ruber TH, obtaining a robust and

119

efficient Rhodococcus biocatalyst for the bio-production of acrylamide.

120

2. Materials and Methods

121

2.1. Strains and media

122

The plasmids and strains used in this study are listed in Table S1 and S2,

123

respectively. E. coli Top10 was used for plasmid construction. R. ruber TH was

124

previously isolated from nitrile-containing soil (Ma et al., 2010) and produced NHase

125

for the conversion of acrylonitrile to acrylamide. Luria-Bertani (LB) medium (10 g/L

126

tryptone, 5 g/L yeast extract, 10 g/L NaCl) was used for the cultivation of E. coli, and

127

seed medium (30 g/L glucose, 1 g/L yeast extract, 7 g/L tryptone, 0.5 g/L

128

K2HPO4•3H2O, 0.5 g/L KH2PO4, 0.5 g/L MgSO4•7H2O, 1 g/L monosodium glutamate,

129

pH 7.5) was used for routine cultivation of R. ruber. When required, 25 µg/mL

130

kanamycin, 6 µg/mL tetracycline or 5 µg/mL chloramphenicol was added. For the

131

overexpression of NHase, a seed culture was transferred to fermentation medium (30

132

g/L glucose, 7–8 g/L yeast extract, 10 g/L urea, 2.28 g/L K2HPO4•3H2O, 0.866 g/L

133

KH2PO4, 1 g/L MgSO4•7H2O, 1 g/L monosodium glutamate, 28.55 mg/L CoCl2•6H2O,

134

pH 7.5).

135

2.2. Construction of plasmids

136

A codon-optimized cas9 gene was amplified from the plasmid pCAS9-mCherry

137

(Addgene #80975) (Schmid-Burgk et al., 2016) with primers XbaI-Cas9-F and

138

BamHI-Cas9-R, and the mcherry gene was amplified with XbaI-mcherry-F and

139

KpnI-mcherry-R. The amplified fragments were inserted into plasmid pNV-Pa2 to

140

construct pNV-Pa2-Cas9 and pNV-Pa2-mcherry, respectively. Plasmid pNV-null-Cas9

141

with no promoter for Cas9 was constructed by inserting cas9 gene into plasmid

142

pNV18.1. For the construction of pNV-Pa2-Cas9::mCherry, cas9 was amplified with

143

XbaI-Cas9-F and Cas9-fusion-R, and the mcherry gene was amplified with

144

fusion-mcherry-F and KpnI-mcherry-R. The amplified fragments were fused together

145

by overlap PCR and inserted into pNV-Pa2. Pa2 was a urea-inducible promoter, and

146

its expression relied on native transcriptional regulators in the genome of R. ruber.

147

pNVTc was a derivative of pNV18.1 generated by replacing the KmR gene with

148

the TcR gene. The backbone of pNV18.1 without the KmR gene was amplified with

149

primers pNV-F and pNV-R, and the TcR gene was amplified from the plasmid pPHA

150

(Ma et al., 2010) with the primers fusion-Tc-F and fusion-Tc-R. The fragments were

151

fused together using Gibson Assembly to obtain pNVTc. Similarly, pNVCm1 and

152

pNVCm were derivatives of pNV18.1 with the KmR gene replaced by the CmR gene.

153

The CmR gene with a native promoter was amplified from pXMJ19 with the primers

154

fusion-Cm-F1 and fusion-Cm-R1 and then fused with the backbone of pNV18.1 to

155

construct pNVCm1. To improve the expression level of CmR in R. ruber, the strong

156

constitutive promoter PamiC (Jiao et al., 2018) from Pa2 was amplified from

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pNV-Pa2 with the primers fusion-PamiC-F and PamiC-R. The CmR gene was

158

amplified from pXMJ19 with the primers fusion-Cm-F and fusion-Cm-R. The

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amplified fragments were fused by overlap PCR and then ligated with the backbone

160

of pNV18.1 using Gibson Assembly to generate pNVCm.

161

Replicons pB264 (Lessard et al., 2004) and pRC4 (Hashimoto et al., 1992) was

162

synthesized by Qinglan (Wuxi, China). pBNVTc was a derivative of pNVTc obtained

163

by replacing the pAL5000 origin with pB264. Plasmid pNVTc and pB264 were both

164

digested by NheI and ligated together to obtain pBNVTc after the pAL5000 origin

165

was removed. Similarly, pRCTc was constructed by replacing the pAL5000 origin of

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pNVTc with pRC4, and pBNVCm was constructed by replacing the pAL5000 origin

167

of pNVCm with pB264.

168

The strong constitutive promoter PamiC, free of ribosome-binding sites, was

169

amplified from pNVCm with the primers KpnI-PamiC-F and PamiC-R1, and the

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sgRNA expression cassette targeting amiE was amplified from pJOE8999

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(Altenbuchner, 2016) with the primers sgRNA-F1 and XbaI-sgRNA-R. The amplified

172

fragments were fused by overlap PCR and inserted into pBNVTc to construct

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pBNVTc-sgRNA1. The upstream homologous arm of amiE was amplified from R.

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ruber TH with the primers XbaI-amiUp-F and amiUp-R, and the downstream arm was

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amplified with amiDown-F and HindIII-amiDown-R. The fragments were fused and

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inserted into pBNVTc-sgRNA1 to construct the pBNVTc-sgRNA1-donor.

177

For the quick and easy assembly of the guide sequence with the sgRNA cassette,

178

a BbsI restriction site was introduced. XbaI-PamiC-F and BbsI-PamiC-R were used to

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amplify PamiC from pNVCm. BbsI-sgRNA-F and KpnI-sgRNA1-R were used to

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amplify the sgRNA cassette from pJOE8999. The fragments were fused and inserted

181

into pBNVCm to construct pBNVCm-BbsI-sgRNA. Afterward, the pBNVCm-sgRNA

182

series could be generated by Golden Gate Assembly as described by Ran et al. (Ran et

183

al., 2013).

184

The Che9c60&61 gene (DeLorenzo et al., 2018) was synthesized by Qinglan

185

(Wuxi, China). The Che9c60&61 gene was amplified with the primers XbaI-che9c-F

186

and KpnI-che9c-R, and promoter Pa2 was amplified from pNV-Pa2 with the primers

187

HindIII-Pa2-F and XbaI-Pa2-R. The two fragments were inserted into pRCTc to

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construct pRCTc-Pa2-Che9c60&61. A homologous arm of amiE was amplified from

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R. ruber with the primers EcoRI-amiE-F and BamHI-amiE-R and inserted into

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pK18mob, a non-replicating plasmid for Rhodococcus, to construct pK18mob-amiE.

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The primers used in this study are listed in Table S3. DNA polymerases,

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restriction enzymes, and T4 DNA ligases were purchased from TaKaRa (Dalian,

193

China) and Vazyme (Nanjing, China). DNA purification kits, gel extraction kits and

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plasmid extraction kits were purchased from Solarbio (Beijing, China). Gibson

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Assembly kits were purchased from Taihe Biotechnology (Beijing, China). DNA

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sequencing was performed by GENEWIZ (Suzhou, China). Plasmids for

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CRISPR/Cas9

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pRCTc-Pa2-Che9c60&61 (Addgene #134766) and pBNVCm-BbsI-sgRNA (Addgene

199

#134767) have been deposited to Addgene (website: http://www.addgene.org/).

200

system

including

pNV-Pa2-Cas9

(Addgene

#134765),

2.3. Transformation protocol

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Plasmids or DNA fragments were introduced into R. ruber by electroporation as

202

described by Jiao et al.(Jiao et al., 2018) with some modifications. R. ruber was

203

cultured in GYKGI media (20 g/L glucose, 5 g/L yeast extract, 0.655 g/L

204

K2HPO4•3H2O, 0.5 g/L KH2PO4, 0.5 g/L MgSO4•7H2O, 8.5 g/L glycine, 1.5 mg/L

205

isoniazid, pH 7.5) at 28°C until OD460 reached 0.3~0.5. The R. ruber cells were

206

collected by centrifugation at 4°C, washed three times with 10% ice-cold glycerol,

207

and finally re-suspended to a 1/200 initial volume with 10% ice-cold glycerol. One

208

microgram of plasmids or DNA fragments were added to 100 µL of competent cells

209

and loaded into an electroporation cuvette with a 2 mm gap. The cuvette was placed

210

in ice for 10 min, and electroporation was performed with a pulse of 12.5 kV/cm. The

211

cells were mixed with 800 µL of LBHIS medium (5 g/L tryptone, 5 g/L NaCl, 2.5 g/L

212

yeast extract, 18.5 g/L brain heart infusion powder and 91 g/L sorbitol) and cultured at

213

28°C for 3 h. The cells were subsequently spread on solid plates (10 g/L glucose, 3

214

g/L yeast extract, 1 g/L NaCl, 2 g/L KH2PO4, 0.2 g/L MgSO4•7H2O, 15 g/L agar) with

215

appropriate antibiotics. Depending on the antibiotics used, the culture time varied

216

from 2 to 5 days.

217

2.4. Growth analysis

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Recombinant cells of R. ruber TH (Cas9) and TH (pNV18.1) were inoculated

219

from solid plates into seed medium and cultured at 28°C and 200 rpm for 48 h. The

220

seed culture was transferred to new seed medium containing 0 g/L or 6 g/L urea to an

221

initial OD460 of 0.3 and cultured at 28°C and 200 rpm. Samples were taken

222

periodically to monitor the growth conditions.

223

2.5. Fluorescence measurement

224

Cells in the mid-exponential growth phase were centrifuged at 4°C and

225

re-suspended in 10 mM PBS buffer to an OD460 of 1.0. The mCherry expression

226

intensities of recombinant cells were measured using a TECAN Infinite M200 PRO

227

microplate reader (Männedorf, Switzerland) with R. ruber TH (pNV18.1) as the

228

negative control. The excitation and emission wavelengths were 535 and 620 nm,

229

respectively.

230

2.6. Determination of transformation efficiency of plasmids derived from E.

231

coli and R. ruber

232

Plasmid pBNVTc-sgRNA1-donor was constructed using E. coli Top 10, and then

233

transformed

to

R.

ruber

TH

by

electroporation.

To

extract

plasmid

234

pBNVTc-sgRNA1-donor from R. ruber, corresponding R. ruber colonies were

235

inoculated to the seed medium, and cultured at 28℃ and 200 rpm for 36 h when OD460

236

reached 3~5. Two microliter of cell liquid were centrifuged at 13000×g for 2 min.

237

Cells were then re-suspended by 2 mL of 15 mg/mL lysozyme solution (dissolved in

238

Tris-EDTA buffer solution), and then incubated at 37℃ and 200 rpm for 1 h. Cells

239

were harvested by centrifugation at 13000×g for 2 min, and then used for plasmid

240

extraction according to the standard protocol of plasmid extraction kits purchased

241

from Solarbio (Beijing, China). About 30 µL plasmid could be obtained with a

242

concentration about 10 ng/µL.

243

The routine extraction of plasmids from E. coli does not require the extra

244

treatment of lysozyme. However, to exclude the influence of lysozyme, we also

245

included this step in the procedure of plasmid extraction from E. coli. About 80 µL

246

plasmid of 200 ng/µL could be obtained from 2 mL cell liquid. The plasmid solution

247

was diluted to 10 ng/µL, the same as the concentration of plasmid derived from R.

248

ruber.

249

To determine the transformation efficiencies of pBNVTc-sgRNA1-donor derived

250

from R. ruber and E. coli, 100 ng plasmids were added to 100 µL of R.ruber

251

competent cells for electroporation. Colonies were counted for the calculation of

252

transformation efficiency.

253

2.7. Characterization of Che9c60&61 in R. ruber

254

R. ruber was transformed with the plasmid pRCTc-Pa2-Che9c60&61. The

255

subsequent strains were transformed with the suicide plasmid pK18mob-amiE or

256

linear homologous arms of amiE flanking the CmR gene. The growing colonies were

257

tested by colony PCR to determine the editing efficiency. The linear CmR integration

258

cassette was prepared as follows. The upstream and downstream homologous arms of

259

amiE were amplified from R. ruber, the CmR cassette was amplified from pNVCm,

260

and the three fragments were fused together by overlap PCR.

261

2.8. Iterative genome editing protocol

262

Wild-type R. ruber was transformed with the plasmid pNV-Pa2-Cas9 and then

263

made competent for the introduction of pRCTc-Pa2-Che9c60&61. The subsequent R.

264

ruber (Cas9+Che9c60&61) was transformed with 1 µg of the pBNVCm-sgRNA

265

series and 1 µg of linear donor DNA and spread on solid plates containing 25 µg/mL

266

kanamycin, 6 µg/mL tetracycline and 5 µg/mL chloramphenicol. The colonies were

267

verified using appropriate primers, and the edited colonies were used for the next

268

round of editing. Before the introduction of new sgRNA plasmids, the previous

269

pBNVCm-sgRNA must be cured. The edited R. ruber was cultured in seed medium

270

containing 25 µg/mL kanamycin and 6 µg/L tetracycline at 37°C before being spread

271

on solid plates containing the above antibiotics. Subsequently, colonies were picked

272

and streaked on plates containing 5 µg/mL chloramphenicol. Colonies not growing on

273

plates

274

pBNVCm-sgRNA and were selected for the next round of gene editing. Similarly,

275

plasmids pNV-Pa2-Cas9 and pRCTc-Pa2-Che9c60&61 could also be cured from the

276

final edited strains when they were cultured in seed medium and streaked on plates

277

without antibiotics.

278

containing

chloramphenicol

were

bacteria

that

had

already

lost

2.9. NHase and amidase activity assay

279

For the overexpression of NHase, a seed culture of R. ruber was transferred to

280

fermentation medium to an initial OD460 of 3.0 and cultured at 28°C for 54 h. For the

281

measurement of NHase activity, 100 µL of free cells (OD460=50) and 4.5 mL of 50

282

mM PBS were mixed with 200 µL of acrylonitrile and incubated at 28°C for 5 min.

283

The reaction was terminated by adding 200 µL of 3 M HCl. After centrifugation at

284

10000×g for 2 min, the supernatant was analyzed with gas chromatography (Chen et

285

al., 2013). One unit of NHase activity was defined as the amount of cells that

286

produced one µmol of acrylamide per minute.

287

For the measurement of amidase activity, 500 µL of free cells (OD460=50) in 50

288

mM PBS was mixed with 500 µL of 20% acrylamide solution and incubated at 40°C

289

for 1 h. The reaction was terminated by centrifugation at 10,000×g for 2 min, and the

290

supernatant was analyzed with gas chromatography. One unit of amidase activity was

291

defined as the amount of cells that converted one µmol of acrylic acid per minute.

292 293

2.10.

Assessment of catalytic stability of R. ruber

The catalytic stability was assessed by measuring the residual NHase activity of

294

R. ruber after a batch of acrylonitrile hydration. The hydration reaction was

295

performed in a 100 mL flask containing 20 mL of free cells in deionized water

296

(OD460=50). Acrylonitrile (15 mL) was added over 40 min at a velocity of 0.375

297

mL/min. The reaction was kept on ice, as it was strongly exothermic. The cells were

298

recovered by centrifugation at 10000×g for 15 min. The supernatant was analyzed

299

using gas chromatography, and the cells were resuspended to 20 mL with deionized

300

water. Then, 100 µL of cell suspension was taken for the measurement of residual

301

NHase activity.

302

2.11.

Multibatch acrylonitrile hydration

303

Multi-batch acrylonitrile hydration was conducted in a 1000 mL flask containing

304

400 mL of R. ruber cell suspension (1.6 gdcw/L). The initial feeding rate of

305

acrylonitrile was set at 5.8 mL/min and adjusted suitably to maintain the acrylonitrile

306

concentration below 1 g/L. The temperature was maintained between 18 and 25°C.

307

The acrylamide concentration was monitored by taking samples periodically for GC

308

analysis. The reaction was terminated when the acrylamide concentration reached 500

309

g/L or did not continue to increase. Afterward, acrylamide was separated from R.

310

ruber cells using a hollow fiber membrane, and the cells were recovered and used for

311

the next batch of acrylonitrile hydration.

312

3. Results

313 314

3.1. Enabling efficient function of the Cas9-sgRNA complex in R. ruber R.

ruber

TH

is

an

organic

solvent-tolerant

strain

isolated

from

315

nitrile-contaminated soil that has been engineered for the expression of nitrile

316

hydratase (Ma et al., 2010), nitrilase (Sun et al., 2016), and epoxide hydrolase (Liang

317

et al., 2019). The proportion of soluble target enzymes in R. ruber reaches 30%~50%

318

of the total protein (Liang et al., 2019; Sun et al., 2016), indicating its potential as an

319

efficient whole-cell biocatalyst. Despite its desirable traits, further engineering of R.

320

ruber has been hindered by its high GC content (~70%) and low transformation and

321

recombination efficiencies. Compared with that of other microorganisms, such as E.

322

coli, C. glutamicum and B. subtilis, the genome editing of Rhodococcus is quite

323

difficult.

324

To develop the CRISPR/Cas9 system in R. ruber, we first needed to enable the

325

Cas9-sgRNA complex to efficiently introduce DSB in the target genomic locus. As

326

the native Streptococcus pyogenes cas9 gene (GC content 35.1%) contains too many

327

extremely rare codons of R. ruber, a codon-optimized cas9 gene(Schmid-Burgk et al.,

328

2016) (GC content 62%) was selected. The results of the codon usage analysis of the

329

cas9 genes are shown in Fig. S1 and S2. Considering the reported toxicity of Cas9 to

330

the cell growth of some bacteria such as C. glutamicum (Cho et al., 2017) and

331

Halomonas (Qin et al., 2018), we chose a urea-inducible promoter with adjustable

332

intensity for the expression of Cas9, Pa2 (Sun et al., 2016), as shown in Fig. 1a. The

333

efficiency of transforming pNV-Pa2-Cas9 into R. ruber was as low as 45±4 cfu/µg

334

DNA. This was probably caused by the large size (8.83 kb) of the plasmid, as the

335

transformation efficiency of plasmid pNV-null-Cas9 with similar size but no promoter

336

for Cas9 was also as low as 53±6 cfu/µg DNA. Consequently, a two-plasmid system

337

with separated Cas9 and sgRNA was designed to validate the function of the

338

Cas9-sgRNA complex. R. ruber TH was transformed with pNV-Pa2-Cas9 and

339

subsequently made competent for the introduction of a pBNVTc-sgRNA series

340

(sgRNA1, sgRNA2 and sgRNA3, targeting genes amiE, nhh, and nit, respectively), in

341

which the strong constitutive promoter PamiC (Jiao et al., 2018), free of

342

ribosome-binding sites, was used to transcribe the sgRNA cassette (Altenbuchner,

343

2016) (Fig. 1b). As shown in Fig. 1c, the efficiency of transforming pBNVTc-sgRNA

344

into R. ruber TH (Cas9) was three orders of magnitude lower than the transformation

345

efficiency for R. ruber TH. The escape rate of R. ruber for Cas9 cleavage was lower

346

than 10-3, indicating that the Cas9-sgRNA complex could function as an efficient

347

counter-selection marker in R. ruber.

348

349 350

Figure 1. Enabling the function of the Cas9-sgRNA complex in R. ruber. (a)

351

Plasmid map of pNV-Pa2-Cas9. (b) Plasmid map of the pBNVTc-sgRNA series. (c)

352

Transformation efficiencies for the pBNVTc-sgRNA series of plasmids into R. ruber

353

TH or TH (Cas9) with the empty pBNVTc as a control. The plasmids

354

pBNVTc-sgRNA1, pBNVTc-sgRNA2, and pBNVTc-sgRNA3 contained sgRNA

355

targeting amiE, nhh, and nit, respectively. The electroporation was performed as

356

described in section 2.3. Urea was not used in this experiment, and the expression of

357

Cas9 relied on the leaky transcription of promoter Pa2. Experiments were performed

358

in triplicate. (d) Illustration of escape mechanisms determined by sequencing the

359

pNV-Pa2-Cas9 and pBNVTc-sgRNA extracted from the escape colonies.

360 361

We also analyzed the colonies escaping from Cas9 cleavage by DNA sequencing.

362

None of the 8 sequenced colonies showed mutations on the target site of sgRNA in

363

the genome, indicating that NHEJ was not observed in these colonies. Although genes

364

such as Ku and LigD (Shuman and Glickman, 2007) responsible for NHEJ could be

365

found in R. ruber (see Fig. S3), the efficiency of NHEJ seemed to be inadequate for

366

the repair of DSBs caused by Cas9. NHEJ pathways in bacteria such Streptomyces

367

coelicolor (Tong et al., 2015) and M. smegmatis (Sun et al., 2018) could repair DSBs

368

when no templates for homologous recombination were present. However, other

369

species such as Clostridium cellulolyticum were killed by Cas9 cleavage despite the

370

existence of NHEJ pathways (Xu et al., 2015). By sequencing the plasmids extracted

371

from these colonies, we found that either sgRNA or Cas9 was inactivated. As shown

372

in Fig. 1d, 6 colonies showed excision of the sgRNA cassette from plasmid

373

pBNVTc-sgRNA, while the others showed a frame shift mutation on Cas9 or insertion

374

by transposases of R. ruber.

375

Considering the possible toxicity of Cas9, we also analyzed its effect on the cell

376

growth of R. ruber. The fluorescent protein mCherry was fused with Cas9 to measure

377

its expression level. The addition of 6 g/L urea improved the expression of

378

Cas9::mCherry by 5.5-fold but showed no influence on the cell growth of R. ruber

379

(see Fig. S4). Unlike its overexpression in some bacteria such as C. glutamicum (Cho

380

et al., 2017)and Halomonas (Qin et al., 2018), the overexpression of Cas9 seemed to

381

be harmless to R. ruber in the absence of sgRNA.

382

3.2. Limited transformation and recombination efficiencies hinder the

383

CRISPR/Cas9-based genome editing of R. ruber

384

As a proof of concept, amiE, which encodes an aliphatic amidase, was selected

385

for gene knockout with CRISPR/Cas9 as shown in Fig. S5. For the repair of DSBs,

386

homologous arms (approximately 800 bp on each side) of amiE were added to the

387

sgRNA plasmid pBNVTc-sgRNA1 to construct the pBNVTc-sgRNA1 donor.

388

However, nearly no colonies were obtained when the pBNVTc-sgRNA1-donor was

389

introduced to R. ruber TH (Cas9) due to the limited transformation efficiency and

390

recombination efficiency of R. ruber.

391

We could estimate the efficiency of genome editing with CRISPR/Cas9 using the

392

following two equations: colony number = T×(R+E) and editing efficiency = R/(R+E),

393

where T represents the transformation efficiency (cfu/µg DNA) of a sgRNA plasmid,

394

R represents the recombination efficiency and E represents the escape rate.

395

For the plasmid pBNVTc-sgRNA1-donor, the homologous arms added to

396

pBNVTc-sgRNA increased the size from 5.52 kb to 7.17 kb, thereby significantly

397

reducing the transformation efficiency of R. ruber from 1.32×104 to 2.06×103 cfu/µg

398

DNA. Considering the limited recombination pathways of most bacteria, it was

399

difficult to obtain enough colonies for the screening of edited colonies with such

400

transformation efficiencies. Thus, to develop an efficient CRISPR/Cas9 system, we

401

needed to improve both the transformation and recombination efficiencies of R. ruber.

402

3.3. Enhancing

transformation

efficiency

through

bypassing

the

403

Rhodococcus restriction-modification system

404

The transformation efficiency of bacteria is affected by multiple factors,

405

including

competent

cell

preparation,

electroporation

parameters

and

406

restriction-modification (RM) systems (Suzuki and Yasui, 2011). Previous

407

CRISPR/Cas9-associated studies in bacteria such as C. glutamicum (Cho et al., 2017)

408

and Saccharopolyspora erythraea (Liu et al., 2018) focused on the first two factors to

409

improve the transformation efficiency. However, in this study of R. ruber,

410

optimization of the competent cell preparation and electroporation (Jiao et al., 2018)

411

did not effectively improve the transformation efficiency as expected. We thus

412

speculated that the RM system in Rhodococcus might significantly reduce their

413

transformation efficiency. To test this possibility, we compared the transformation

414

efficiency of shuttle vectors extracted from E. coli and R. ruber. As shown in Fig. 2a,

415

the transformation efficiency of plasmid pBNVTc-sgRNA1-donor extracted from R.

416

ruber was 89-fold higher than that extracted from E. coli. We performed Sanger

417

sequencing to the two plasmids but they showed no difference in DNA sequence (see

418

Fig. S6). However, according to Pacbio single-molecule, real-time (SMRT)

419

sequencing data submitted to the Restriction Enzyme Database (REBASE), the

420

methylation patterns in R. ruber and E. coli were quite different as summarized in

421

Table S4. Thus we speculated that the modification status of the plasmids affected

422

their transformation efficiencies to Rhodococccus. That is, the shuttle vectors

423

extracted from E. coli in a routine method might be digested by the restriction

424

enzymes of the target hosts, thereby reducing the transformation efficiency. However,

425

vectors extracted from the target hosts could be methylated by modification systems

426

and avoid digestion when transformed again into the hosts (Fig. 2b). Based on this

427

idea, we proposed a simple but effective strategy to improve the transformation

428

efficiency through bypassing the RM system. In the CRISPR/Cas9 system, the

429

plasmid pBNVTc-sgRNA1-donor could be transformed to R. ruber TH for

430

pre-methylation and then extracted and re-transformed into R. ruber TH(Cas9) to

431

obtain enough colonies for gene knockout.

432

433 434

Figure 2. Enhancing transformation efficiency through bypassing the RM system.

435

(a) The effect of the RM system on the transformation efficiency of the plasmid

436

pBNVTc-sgRNA1-donor. The plasmid was extracted from E. coli and R. ruber and

437

transformed into R. ruber to estimate the efficiency. Experiments were performed in

438

triplicate. (b) Schematic diagram illustrating the bypass of the restriction-modification

439

system.

440

3.4. Enhancing recombination efficiency by introducing heterologous

441

recombinases Che9c60 and Che9c61 and linear donor DNA

442

For the amiE gene knockout, the plasmid pBNVTc-sgRNA1-donor was extracted

443

from R. ruber and transformed into R. ruber TH (Cas9), and 136 colonies were

444

obtained. However, only 1 of 96 colonies picked was amiE-negative, as shown in Fig.

445

3. To improve the editing efficiency, we tried various methods, including the addition

446

of a linear donor and optimization of donor length, but no significant difference was

447

observed. The results indicated that the endogenous recombination pathways of R.

448

ruber were insufficient for the repair of DSBs.

449 450

Figure 3. Gene knockout of amiE with the two-plasmid CRISPR/Cas9 system.

451

The plasmid pBNVTc-sgRNA1-donor was extracted from R. ruber and transformed

452

into R. ruber TH (Cas9) for gene knockout. Primers P1 and P2 were used to examine

453

the edited colonies. A fragment of 1426 bp was amplified from the edited colonies,

454

and a fragment of 2464 bp was amplified from the wild type. Results from 23 of the

455

96 colonies tested are displayed.

456 457

To improve the editing efficiency, exogenous recombination systems are often

458

required to facilitate homologous recombination (Cho et al., 2017; DeLorenzo et al.,

459

2018; Oh and van Pijkeren, 2014; Wang et al., 2018) . In this study, the bacteriophage

460

recombinases Che9c60 and Che9c61 (van Kessel and Hatfull, 2007) were introduced

461

and characterized in R. ruber. Recombinases Che9c60 and Che9c61 are GC-rich

462

homologs of RecE and RecT, respectively. Che9c60 is an exonuclease that produces a

463

single-stranded DNA (ssDNA), and Che9c61 is a ssDNA-binding protein that

464

facilitates strand invasion and exchange of the single-stranded integration cassette into

465

the targeted DNA (van Kessel and Hatfull, 2007). Before incorporating Che9c60&61

466

into the two-plasmid CRISPR/Cas9 system, we first tested whether they could

467

facilitate the homologous recombination of donor DNA supplied on circular plasmids.

468

The plasmid pRCTc-Pa2-Che9c60&61 was constructed with Che9c60&61 driven by

469

the urea-induced promoter Pa2 and then transformed into R. ruber. Subsequently, the

470

non-replicating plasmid pK18mob-amiE containing a homologous sequence of amiE

471

or linear homologous arms of amiE flanking a chloramphenicol resistance (CmR)

472

cassette, was introduced to validate the function of Che9c60&61 in R. ruber. The

473

schematic diagrams of homologous recombination are shown in Fig. S7, and the

474

results are summarized in Table 1.

475

Table 1 Gene knockout of amiE using a suicide plasmid or linear fragment by

476

homologous recombination assisted or not assisted by recombinases Host strains Donors

R. ruber TH

R. ruber TH (Che9c60&61)

Plasmid

Linear

Plasmid

Linear

pK18mob-amiE

amiE-CmR

pK18mob-amiE

amiE-CmR

Colonies

5

0

3

196

Editing efficiency

0/5

-

0/3

8/8

477

Note: Plasmid pK18mob-amiE was a non-replicating plasmid containing a

478

homologous sequence (785 bp) of the gene amiE, and amiE-CmR was a linear

479

integration cassette with the upstream (750 bp) and downstream (750 bp) homologous

480

arms of amiE flanking the CmR gene.

481 482

For the non-replicating plasmid pK18mob-amiE, only 5 and 3 colonies harboring

483

the KmR gene were obtained from R. ruber TH and R. ruber TH (Che9c60&61),

484

respectively, but none of them were amiE-negative regardless of whether

485

Che9c60&61 was used. The results confirmed the severe illegitimate recombination

486

in R. ruber, which caused a low efficiency for gene editing. For linear donor DNA

487

containing the upstream and downstream homologous arms of amiE flanking the CmR

488

gene, no colonies were obtained from R. ruber TH, while 196 colonies were obtained

489

from R. ruber TH (Che9c60&61) with an editing efficiency of 100%. That is,

490

Che9c60&61 can remarkably facilitate the homologous recombination of linear donor

491

DNA in R. ruber over that of plasmid, and the recombination efficiency was

492

successfully enhanced by coupling the introduction of heterologous recombinases

493

Che9c60&61 and linear donor dsDNA.

494

3.5. A triple-plasmid CRISPR/Cas9 recombineering system for genome

495

editing of R. ruber

496

To further incorporate Che9c60&61 into the CRISPR/Cas9 system, we first

497

constructed a plasmid harboring Cas9 and Che9c60&61 but failed to transform it into

498

R. ruber due to its size being too large (11.3 kb). A triple-plasmid system was thus

499

developed as shown in Fig. 4. The plasmid pNV-Pa2-Cas9, expressing Cas9 protein,

500

was first transformed into R. ruber, obtaining the engineered strain R. ruber TH

501

(Cas9).

502

Che9c60&61, was subsequently transformed into TH (Cas9), resulting in the second

503

engineered strain TH (Cas9+Che9c60&61). Then, competent cells of TH

504

(Cas9+Che9c60&61) were prepared and used for co-transformation of the third

505

plasmid containing sgRNA and the linear donor DNA containing the upstream and

506

downstream sequences of the target gene.

507

The

plasmid

pRCTc-Pa2-Che9c60&61,

expressing

recombinases

508

Figure 4. Triple-plasmid CRISPR/Cas9-mediated recombineering system for the

509

genome editing of R. ruber. Plasmids pNV-Pa2-Cas9 and pRCTc-Pa2-Che9c60&61

510

were transformed to R. ruber first, and the subsequent strain was made competent for

511

the introduction of the pBNVCm-sgRNA series and linear donor dsDNA.

512 513

Here, a temperature-sensitive replicon, pB264 (Lessard et al., 2004), was

514

specifically used for the expression of the sgRNA cassette. A CmR gene with its native

515

promoter was first amplified from pXMJ19 to construct the E. coli-Rhodococcus

516

shuttle vector pBNVCm1, but the cultivation time of R. ruber had to be lengthened

517

from 2~3 days to 5~6 days. To solve this problem, the strong constitutive promoter

518

PamiC (Jiao et al., 2018) was utilized to improve the expression level of the CmR

519

gene, and the transformation efficiency of the new plasmid, pBNVCm, into R. ruber

520

reached 4.05×104 cfu/µg DNA in 3 days. For the quick assembly of the guide

521

sequence to sgRNA, a BbsI restriction site was introduced to construct

522

pBNVCm-BbsI-sgRNA (see Fig. S8). In this way, the guide sequences of target genes

523

could be assembled into pBNVCm-BbsI-sgRNA quickly and conveniently via Golden

524

Gate Assembly as described by Ran et al. (Ran et al., 2013).

525

3.6. Gene knockout, insertion and mutation efficiencies of R. ruber with the

526

triple-plasmid CRISPR/Cas9 recombineering system

527

As a proof of concept, the plasmid pBNVCm-sgRNA1 and the linear

528

homologous arms of amiE were co-transformed into R. ruber TH (Cas9+Che9c60&61)

529

for the knockout of amiE. As shown in Fig. 5a, the editing efficiency reached 12/16

530

(75%). This CRISPR/Cas9-based recombineering system was used to successfully

531

knock out several other genes, as summarized in Table 2.

532 533

Table 2 Genome editing with CRISPR/Cas9-based recombineering system Gene editing

Guide sequencea

Deletion

Editing

length (bp)

efficiencyb

534 535 536

amiE deletion

GTGATCCTCATGGCGCTCGC

1038

12/16

nhh deletion

CCCGGGACGGTCTGCGCCCA

1314

2/3

for deletion

AGGAACTCGGCCACAACCGG

1275

4/8

ami deletion

GACCATCCCCGGCGACGAAA

1037

5/24

nit deletion

CGGCATGACCGGATACGGAC

2858

1/32

gfp insertion

GTGATCCTCATGGCGCTCGC

-

1/4

nhh mutation

CCCGGGACGGTCTGCGCCCA

-

20/33

a. The guide sequence was designed using a web-based tool developed by Park et al. (Park et al., 2015) b. Number of edited colonies/total number of transformants.

537 538

Gene insertion and gene mutation were also tested with this system, as shown in

539

Fig. 5b and 5c. The plasmid pBNVCm-sgRNA1 and homologous arms of amiE

540

flanking a gfp gene were co-transformed into R. ruber TH (Cas9+Che9c60&61), and

541

the gene insertion efficiency was 1/4 (25%). Only a minor modification was

542

sometimes needed for the target gene, so the efficiency of introducing in situ point

543

mutations was tested with this system. The homologous arms of nhh flanking a

544

mutated nhh gene were co-transformed with pBNVCm-sgRNA2 into R. ruber TH

545

(Cas9+Che9c60&61). The NGG for the guide sequence on the wild-type nhh gene

546

was changed to NCG on the mutated sequence, so the edited sequence would avoid

547

cleavage by Cas9. The editing efficiency for a single point mutation reached 20/33

548

(60%). The results demonstrated that the CRISPR/Cas9-based recombineering system

549

was efficient for gene knockout, insertion and mutation.

550

551 552

Figure 5. Gene knockout, insertion and mutation using the CRISPR/Cas9 system.

553

(a) Deletion of the amiE gene encoding amidase. Primers P1 and P2 were used to

554

examine the edited colonies. A fragment of 1426 bp was amplified from the edited

555

colonies, and a fragment of 2464 bp was amplified from the wild type. (b) Insertion of

556

a gfp gene. The expression of GFP was visually identified by a Nikon Eclipse Ti-E

557

inverted fluorescence microscope. (d) Mutation of the nhh gene encoding nitrile

558

hydratase (NHase). The mutation was confirmed by Sanger sequencing.

559

3.7. Iterative gene-editing protocol with the CRISPR/Cas9 system

560

As the CRISPR/Cas9-based recombineering was successful for the knockout of a

561

single gene, we further aimed to develop it for multiple gene editing, which requires

562

an iterative protocol. For this process, the sgRNA plasmid in the edited cells must be

563

cured before the introduction of the next sgRNA plasmid. As stated above, the

564

replicon pB264 for the pBNVCm-sgRNA series was temperature-sensitive and could

565

be cured when the cells were cultured at 37°C. As shown in Fig. 6, the edited cells

566

harboring three plasmids could be cultured at 37°C without chloramphenicol for the

567

curing of pBNVCm-sgRNA, and the loss rate of the plasmid reached 100% (see Fig.

568

S9). The subsequent cells could be made competent for the next round of gene

569

editing.

570

To obtain the final edited strains, all three plasmids were simultaneously cured

571

with culturing at 37°C without any antibiotics. For plasmids pNV-Pa2-Cas9 and

572

pRCTc-Pa2-Che9c60&61, although no temperature-sensitive version of the replicons

573

was available, the two plasmids were unstable in the absence of antibiotics probably

574

due to the burden caused by Cas9 and Che9c60&61. The simultaneous curing

575

efficiency of the three plasmids reached 25% (see Fig. S9), which was acceptable for

576

the screening of plasmid-free edited strains.

577

578 579

Figure 6. Scheme of iterative genome editing in R. ruber. When a round of

580

genome editing finished, the plasmid pBNVCm-sgRNA in the edited cells could be

581

cured at 37°C with Tc and Km but without Cm, and the subsequent strain would be

582

used for the next round of editing. Three plasmids in the final edited strains could also

583

be cured simultaneously by culturing at 37°C without any antibiotics.

584

3.8. Engineering a R. ruber whole-cell biocatalyst for acrylamide production

585

using the novel CRISPR/Cas9 tool

586

As a proof-of-concept example, we applied the CRISPR/Cas9 system in the

587

engineering of R. ruber as a whole cell biocatalyst for the bio-production of

588

acrylamide from acrylonitrile. The metabolic pathways of acrylonitrile in R. ruber are

589

shown in Fig. 7a. R. ruber harboring nitrile hydratase (NHase) has been successfully

590

applied in the bio-production of acrylamide from acrylonitrile. However, the substrate

591

acrylonitrile could be converted by nitrilase into acrylic acid and the product

592

acrylamide could also be further catalyzed by amidase into acrylic acid, resulting in

593

reduced acrylamide yield and increased separation cost. NHase has been cloned in

594

some host strains free of nitrile metabolism, such as E. coli (Chen et al., 2013) and C.

595

glutamicum (Kang et al., 2014), but their application was limited by low enzyme

596

stability or activity. Therefore, native NHase-producing Rhodococcus strains are still

597

preferred for industrial-scale production of acrylamide.

598

To address the problem of byproduct formation, related genes in R. ruber TH

599

were analyzed. One gene of R. ruber TH was predicted to be nitrilase, and 24 genes

600

were predicted to be amidases (See Table S5). As nitrilase and NHase compete for the

601

same substrate (acrylonitrile), it was difficult to measure the nitrilase activity of

602

wild-type Rhodococcus. Thus, a NHase-negative Rhodococcus was constructed, and it

603

showed no nitrilase activity toward acrylonitrile as shown in Fig. S10, indicating that

604

in R. ruber

605

formation. Among the 24 predicted amidases, one aliphatic amidase gene (amiE)

606

reported by Ma et al. (Ma et al., 2010) was selected and knocked out, reducing the

607

amidase activity of R. ruber by 60%, as shown in Fig. 7b and 7c.

amidases rather than a nitrilase were responsible for byproduct

608

The enhancement of the enzyme stability of NHase is also urgently needed owing

609

to the inactivation caused by harsh conditions, including the strong exothermic

610

reaction of acrylonitrile to acrylamide and the high toxicity of polar organic solvents.

611

Previously, a mutant NHase was obtained in our laboratory and showed enhanced

612

stability during acrylonitrile hydration (Yu et al., 2017), but plasmid-based expression

613

of the mutant NHase in R. ruber was affected by the natively overexpressed wild-type

614

NHase and plasmid stability. Thus, based on the amiE-negative R. ruber strain TH△

615

amiE, we performed an iterative procedure to replace the wild-type NHase with the

616

mutant and obtained R. ruber THY. The NHase activity of the engineered strain THY

617

(4976±209 U/mL) showed no significant difference from that of TH and TH△amiE

618

(see Fig. S11), but its catalytic stability was enhanced significantly. The activity

619

retained by THY after one batch of acrylonitrile hydration was 62%, while that

620

retained by R. ruber TH△amiE was only 28%, as shown in Fig. 7d.

621

R. ruber THY was then utilized for multi-batch bio-production of high

622

concentrations of acrylamide from acrylonitrile. After 2.7 h of acrylonitrile hydration

623

using free cells of THY (1.6 gdcw/L) as biocatalyst, the concentration of acrylamide

624

reached 500 g/L with a byproduct acrylic acid concentration as low as 0.5 g/L, and the

625

cells could be recycled 4 times, as shown in Fig. 7e and 7f. Compared with the results

626

using wild-type R. ruber TH, the formation of the byproduct acrylic acid was reduced

627

by 80%, the final concentration of acrylamide was improved by 23.5%, and the

628

number of times that the cells could be recycled was enhanced by 3-fold.

629

630 631

Figure 7. Applying CRISPR/Cas9-based recombineering to engineer R. ruber for

632

the bio-production of acrylamide. (a) Acrylonitrile-degradation pathways in R. ruber.

633

(b) Iterative genome editing to delete one amidase gene and replace the wild-type

634

NHase with a mutant NHase. (c) Amidase activity of the engineered R. ruber. (d)

635

Residual NHase activity of engineered R. ruber after a batch of acrylonitrile hydration.

636

(e) Multi-batch acrylonitrile hydration using R. ruber as biocatalyst. (f) Acrylic acid

637

concentration during the first batch of acrylonitrile hydration.

638

4. Discussion

639

The CRISPR/Cas9 system has emerged as a powerful genome editing tool in

640

animals, plants, and microorganisms. Nevertheless, this system has not been applied

641

in some non-model strains with desirable traits but lacking of genetic tractability.

642

Rhodococcus spp. are known as organic solvent-tolerant strains with diverse

643

metabolic pathways and play an important role in bioremediation, biotransformation,

644

and bioconversion (Bell et al., 1998). For example, R. ruber TH has been engineered

645

as a robust and efficient whole-cell biocatalyst for the overexpression of NHase,

646

nitrilase and epoxide hydrolase (Liang et al., 2019; Ma et al., 2010; Sun et al., 2016).

647

Their desirable traits make Rhodococcus a promising platform for biotransformation,

648

which often involves harsh conditions such as toxic organic solvents. However,

649

genome editing of Rhodococcus is still difficult due to the low transformation and

650

recombination efficiencies. In this study, we adopted strategies to overcome the two

651

bottlenecks and developed an efficient CRISPR/Cas9-mediated method in R. ruber

652

for gene knockout, insertion and mutation.

653

The transformation efficiency determines whether enough colonies could be

654

obtained for the screening of mutants. Transformation is affected by multiple factors,

655

including the physical barrier of cell surface structures, electric stress during

656

electroporation, and the RM system of the target host (Suzuki and Yasui, 2011).

657

Previous studies to develop a CRISPR system in bacteria mainly focused on the

658

optimization of competent cell preparation and electroporation to enhance the

659

transformation efficiency (Cho et al., 2017; Liu et al., 2018). However, for R. ruber,

660

the RM system was the critical barrier hindering the transformation of plasmids.

661

Bypassing the RM system improved the transformation efficiency of R. ruber by

662

89-fold and made the screening of edited colonies feasible. Nevertheless, the method

663

required the extraction of the plasmid pBNVTc-sgRNA1-donor from R. ruber TH,

664

which was time-consuming and laborious. Some more convenient methods to bypass

665

RM systems have been reported, including the knockout of key restriction enzymes of

666

the target host (Holt et al., 2012), in vitro methylation of plasmids (Zhao et al., 2018),

667

and the construction of engineered E. coli hosts for plasmid artificial modification

668

(Suzuki and Yasui, 2011). For further research, restriction enzymes should be

669

analyzed and knocked out to construct a mutant strain with high transformation

670

efficiency, or an effective modification method should be established for R. ruber.

671

The recombination efficiency determines the ratio of edited colonies to the

672

overall number of colonies obtained and is also called the editing efficiency. Due to

673

the limitations of endogenous recombination pathways in most bacteria, exogenous

674

recombination systems such as λ-Red and RecET are often required to facilitate

675

genome editing in E. coli (Jiang et al., 2015), C. gultamicum (Wang et al., 2018), and

676

Lactobacillus reuteri (Oh and van Pijkeren, 2014). Previously, a pair of bacteriophage

677

recombinases, Che9c60&61, were introduced into R. opacus PD630 and

678

constitutively expressed for gene knockout with a linear antibiotic integration

679

cassette(DeLorenzo et al., 2018). In this work, we successfully introduced

680

Che9c60&61 into R. ruber and overcame the low recombination efficiency of R.

681

ruber, improving the editing efficiency of the CRISPR/Cas9 system from 1% to 75%.

682

The establishment of the CRISPR/Cas9 system has expanded our ability to

683

engineer Rhodococcus to meet the requirements of industrial applications. Previously,

684

only

685

recombineering using a linear integration cassette were available for the genome

686

editing of Rhodococcus. However, the former is affected greatly by illegitimate

687

recombination in Rhodococcus (Desomer et al., 1991), while the latter is limited by

688

the recovery of antibiotic cassettes when multiple rounds of editing are needed. With

689

the CRISPR/Cas9 system developed in this study, we achieved efficient and scarless

690

gene knockout and mutation. As an example, R. ruber was engineered as a robust

691

whole-cell biocatalyst for the production of acrylamide, an important bulk chemical

692

whose polymer is widely used for enhanced oil recovery and water treatment (Ma et

693

al., 2010). The industrial bio-production of acrylamide has suffered from the

694

byproduct formation caused by the competing pathways such as amidase and nitrilase,

695

as well as the insufficient enzyme stability of NHase. Iteratively knocking out the

696

critical byproduct-related amidase gene and replacing the wild-type NHase in the

697

genome with a stable mutant with the CRISPR/Cas9 system reduced the byproduct

698

formation by 80%, improved the acrylamide concentration by 23.5%, and enhanced

traditional

homologous

recombination

using

suicide

plasmids

and

699

the number of times that the cells could be recycled by 3-fold. With the knockout of

700

the competing pathway and the enhancement of the target pathway, the engineered

701

strain showed great potential for industrial-scale production of acrylamide with

702

reduced energy and material consumption.

703

In summary, a CRISPR/Cas9-based recombineering system was developed in R.

704

ruber for efficient and scarless genome editing, including gene deletion, insertion and

705

mutation. The genome editing tool was successfully utilized to engineer the organic

706

solvent-tolerant strain R. ruber as a robust and efficient whole-cell biocatalyst with

707

reduced byproduct formation and enhanced catalytic stability for the bio-production

708

of acrylamide. It is expected that through some modifications, the system will be

709

extended to other Rhodococcus strains.

710 711

Acknowledgments: We thank Professor Jun Ishikawa (Japan) for supplying plasmid

712

pNV18.1.

713

Funding: This work was supported by the National Natural Science Foundation of

714

China (No. 21776157; No. 21476126).

715

Conflict of Interest: The authors declare that they have no conflict of interest.

716

References

717

Altenbuchner, J., 2016. Editing of the Bacillus subtilis Genome by the CRISPR-Cas9

718 719 720

System. Appl Environ Microb. 82, 5421-5427. Bell, K. S., Philp, J. C., Aw, D. W. J., Christofi, N., 1998. A review - The genus Rhodococcus. J Appl Microbiol. 85, 195-210.

721

Chen, J., Yu, H. M., Liu, C. C., Liu, J., Shen, Z. Y., 2013. Improving stability of nitrile

722

hydratase by bridging the salt-bridges in specific thermal-sensitive regions. J

723

Biotechnol. 164, 354-362.

724

Cho, J. S., Choi, K. R., Prabowo, C. P. S., Shin, J. H., Yang, D., Jang, J., Lee, S. Y.,

725

2017. CRISPR/Cas9-coupled recombineering for metabolic engineering of

726 727 728

Corynebacterium glutamicum. Metab Eng. 42, 157-167. Choi, K. R., Lee, S. Y., 2016. CRISPR technologies for bacterial systems: Current achievements and future directions. Biotechnol Adv. 34, 1180-1209.

729

Cobb, R. E., Wang, Y., Zhao, H., 2015. High-efficiency multiplex genome editing of

730

Streptomyces species using an engineered CRISPR/Cas system. ACS Synth

731

Biol. 4, 723-8.

732

Crouzet, J., Naudin, L., Orsini, C., Vigne, E., Ferrero, L., LeRoux, A., Benoit, P.,

733

LAtta, M., Torrent, C., Branellec, D., Denefle, P., Mayaux, J. F., Perricaudet,

734

M., Yeh, P., 1997. Recombinational construction in Escherichia coli of

735

infectious adenoviral genomes. P Natl Acad Sci USA. 94, 1414-1419.

736

DeLorenzo, D. M., Henson, W. R., Moon, T. S., 2017. Development of Chemical and

737

Metabolite Sensors for Rhodococcus opacus PD630. ACS Synth Biol. 6,

738

1973-1978.

739

DeLorenzo, D. M., Rottinghaus, A. G., Henson, W. R., Moon, T. S., 2018. Molecular

740

Toolkit for Gene Expression Control and Genome Modification in

741

Rhodococcus opacus PD630. ACS Synth Biol. 7, 727-738.

742

Desomer, J., Crespi, M., Vanmontagu, M., 1991. Illegitimate Integration of

743

Non-Replicative Vectors in the Genome of Rhodococcus-Fascians Upon

744

Electrotransformation as an Insertional Mutagenesis System. Mol Microbiol. 5,

745

2115-2124.

746

DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., Church, G. M., 2013.

747

Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.

748

Nucleic Acids Res 41, 4336-4343.

749

Duran, R., 1998. New shuttle vectors for Rhodococcus sp. R312 (formerly

750

Brevibacterium sp. R312), a nitrile hydratase producing strain. J Basic Microb.

751

38, 101-106.

752

Hashimoto, Y., Nishiyama, M., Yu, F., Watanabe, I., Horinouchi, S., Beppu, T., 1992.

753

Development of a Host Vector System in a Rhodococcus Strain and Its Use for

754

Expression of the Cloned Nitrile Hydratase Gene-Cluster. J Gen Microbiol.

755

138, 1003-1010.

756

Henson, W. R., Campbell, T., DeLorenzo, D. M., Gao, Y., Berla, B., Kim, S. J., Foston,

757

M., Moon, T. S., Dantas, G., 2018. Multi-omic elucidation of aromatic

758

catabolism in adaptively evolved Rhodococcus opacus. Metab Eng. 49, 69-83.

759

Holt, J. P., Grant, A. J., Coward, C., Maskell, D. J., Quinlan, J. J., 2012. Identification

760

of Cj1051c as a major determinant for the restriction barrier of Campylobacter

761

jejuni strain NCTC11168. Appl Environ Microbiol. 78, 7841-8.

762

Jiang, Y., Chen, B., Duan, C. L., Sun, B. B., Yang, J. J., Yang, S., 2015. Multigene

763

Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Appl

764

Environ Microb. 81, 2506-2514.

765

Jiao, S., Yu, H. M., Shen, Z. Y., 2018. Core element characterization of Rhodococcus

766

promoters and development of a promoter-RBS mini-pool with different

767

activity levels for efficient gene expression. New Biotechnol. 44, 41-49.

768

Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., Charpentier, E., 2012.

769

A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive

770

Bacterial Immunity. Science. 337, 816-821.

771

Kang, M. S., Han, S. S., Kim, M. Y., Kim, B. Y., Huh, J. P., Kim, H. S., Lee, J. H.,

772

2014. High-level expression in Corynebacterium glutamicum of nitrile

773

hydratase from Rhodococcus rhodochrous for acrylamide production. Appl

774

Microbiol Biot. 98, 4379-4387.

775

Lessard, P. A., O'Brien, X. M., Currie, D. H., Sinskey, A. J., 2004. pB264, a small,

776

mobilizable, temperature sensitive plasmid from Rhodococcus. BMC

777

Microbiol. 4.

778

Liang, Y., Jiao, S., Wang, M., Yu, H., Shen, Z., 2019. Overexpression of epoxide

779

hydrolase in Rhodococcus ruber with high robustness for the synthesis of

780

chiral epichlorohydrin. Process Biochem. 79, 49-56.

781

Liu, Y., Wei, W. P., Ye, B. C., 2018. High GC Content Cas9-Mediated

782

Genome-Editing

and

Biosynthetic

Gene

Cluster

783

Saccharopolyspora erythraea. ACS Synth Biol. 7, 1338-1348.

Activation

in

784

Ma, Y. C., Yu, H. M., Pan, W. Y., Liu, C. C., Zhang, S. L., Shen, Z. Y., 2010.

785

Identification of nitrile hydratase-producing Rhodococcus ruber TH and

786

characterization of an amiE-negative mutant. Bioresource Technol. 101,

787

285-291.

788

MethCohn, O., Wang, M. X., 1997. An in-depth study of the biotransformation of

789

nitriles into amides and/or acids using Rhodococcus rhodochrous AJ270. J

790

Chem Soc Perk T 1. 1099-1104.

791

Nagasawa, T., Shimizu, H., Yamada, H., 1993. The Superiority of the 3rd-Generation

792

Catalyst,

793

Industrial-Production of Acrylamide. Appl Microbiol Biot. 40, 189-195.

794

Oh, J. H., van Pijkeren, J. P., 2014. CRISPR-Cas9-assisted recombineering in

795 796 797

Rhodococcus-Rhodochrous

J1

Nitrile

Hydratase,

for

Lactobacillus reuteri. Nucleic Acids Res. 42, e131. Park, J., Bae, S., Kim, J. S., 2015. Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics. 31, 4014-4016.

798

Pelicic, V., Reyrat, J. M., Gicquel, B., 1996. Expression of the Bacillus subtilis sacB

799

gene confers sucrose sensitivity on mycobacteria. J Bacteriol. 178, 1197-1199.

800

Qin, Q., Ling, C., Zhao, Y., Yang, T., Yin, J., Guo, Y., Chen, G. Q., 2018.

801

CRISPR/Cas9 editing genome of extremophile Halomonas spp. Metab Eng.

802

47, 219-229.

803 804

Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., Zhang, F., 2013. Genome engineering using the CRISPR-Cas9 system. Nature Protoc. 8, 2281-2308.

805

Schafer, A., Tauch, A., Jager, W., Kalinowski, J., Thierbach, G., Puhler, A., 1994.

806

Small Mobilizable Multipurpose Cloning Vectors Derived from the

807

Escherichia-Coli Plasmids Pk18 and Pk19 - Selection of Defined Deletions in

808

the Chromosome of Corynebacterium-Glutamicum. Gene. 145, 69-73.

809

Schmid-Burgk, J. L., Honing, K., Ebert, T. S., Hornung, V., 2016. CRISPaint allows

810

modular base-specific gene tagging using a ligase-4-dependent mechanism.

811

Nature Commun. 7.

812

Sekizaki, T., Tanoue, T., Osaki, M., Shimoji, Y., Tsubaki, S., Takai, S., 1998.

813

Improved electroporation of Rhodococcus equi. J Vet Med Sci. 60, 277-279.

814

Shao, Z., Dick, W. A., Behki, R. M., 1995. An Improved Escherichia-Coli

815

Rhodococcus Shuttle Vector and Plasmid Transformation in Rhodococcus Spp

816

Using Electroporation. Lett Appl Microbiol. 21, 261-266.

817

Shi, T., Wang, G. L., Wang, Z. W., Fu, J., Chen, T., Zhao, X. M., 2013. Establishment

818

of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a

819

Double-Strand Break in the Chromosome. Plos One. 8.

820 821

Shuman, S., Glickman, M. S., 2007. Bacterial DNA repair by non-homologous end joining. Nat Rev Microbiol. 5, 852-61.

822

Sun, B., Yang, J., Yang, S., Ye, R. D., Chen, D., Jiang, Y., 2018. A

823

CRISPR-Cpf1-Assisted Non-Homologous End Joining Genome Editing

824

System of Mycobacterium smegmatis. Biotechnol J. 13, e1700588.

825

Sun, J. Z., Yu, H. M., Chen, J., Luo, H., Shen, Z. Y., 2016. Ammonium acrylate

826

biomanufacturing by an engineered Rhodococcus ruber with nitrilase

827

overexpression and double-knockout of nitrile hydratase and amidase. J Ind

828

Microbiol Biot. 43, 1631-1639.

829

Suzuki, T., Yasui, K., 2011. Plasmid Artificial Modification: A Novel Method for

830

Efficient DNA Transfer into Bacteria. Methods Mol Biol. 765, 309-326.

831

Tong, Y., Charusanti, P., Zhang, L., Weber, T., Lee, S. Y., 2015. CRISPR-Cas9 Based

832

Engineering of Actinomycetal Genomes. ACS Synth Biol. 4, 1020-9.

833

van der Geize, R., de Jong, W., Hessels, G. I., Grommen, A. W., Jacobs, A. A.,

834

Dijkhuizen, L., 2008. A novel method to generate unmarked gene deletions in

835

the

836

conditional lethality. Nucleic Acids Res. 36, e151.

intracellular

pathogen

Rhodococcus

equi

using

5-fluorocytosine

837

van der Geize, R., Hessels, G. I., van Gerwen, R., van der Meijden, P., Dijkhuizen, L.,

838

2001. Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid

839

Delta(1)-dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as

840

counter-selectable marker. Fems Microbiol Lett. 205, 197-202.

841

van Kessel, J. C., Hatfull, G. F., 2007. Recombineering in Mycobacterium

842

tuberculosis. Nat Methods. 4, 147-52.

843

Wang, B., Hu, Q. T., Zhang, Y., Shi, R. L., Chai, X., Liu, Z., Shang, X. L., Zhang, Y.,

844

Wen, T. Y., 2018. A RecET-assisted CRISPR-Cas9 genome editing in

845

Corynebacterium glutamicum. Microb Cell Fact. 17.

846 847 848 849 850 851

Wang, H. F., La Russa, M., Qi, L. S., 2016. CRISPR/Cas9 in Genome Editing and Beyond. Annu Rev Biochem. 85, 227-264. Wang, M. X., 2015. Enantioselective Biotransformations of Nitriles in Organic Synthesis. Accounts Chem Res. 48, 602-611. Warhurst, A. M., Fewson, C. A., 1994. Biotransformations Catalyzed by the Genus Rhodococcus. Crit Rev Biotechnol. 14, 29-73.

852

Xu, R., Zhang, K., Liu, P., Han, H. W., Zhao, S., Kakade, A., Khan, A., Du, D. L., Li,

853

X. K., 2018. Lignin depolymerization and utilization by bacteria. Bioresource

854

Technol. 269, 557-566.

855

Xu, T., Li, Y., Shi, Z., Hemme, C. L., Li, Y., Zhu, Y., Van Nostrand, J. D., He, Z.,

856

Zhou, J., 2015. Efficient Genome Editing in Clostridium cellulolyticum via

857

CRISPR-Cas9 Nickase. Appl Environ Microbiol. 81, 4423-31.

858 859

Yu, H., Jiao, S., Zhang, j., Shen, Z., An engineered nitrile hydratase and its application. Vol. CN107177581A, China, 2017.

860

Zhang, X. Z., Yan, X., Cui, Z. L., Hong, Q., Li, S. P., 2006. mazF, a novel

861

counter-selectable marker for unmarked chromosomal manipulation in

862

Bacillus subtilis. Nucleic Acids Res. 34.

863

Zhao, H., Xu, L., Rong, Q., Xu, Z., Ding, Y., Zhang, Y., Wu, Y., Li, B., Ji, X., 2018.

864

Application of methylation in improving plasmid transformation into

865

Helicobacter pylori. J Microbiol Methods. 150, 18-23.

866

Highlights Bypassing RM system of Rhodococcus enhanced the transformation efficiency by 89-fold. Introducing bacteriophage recombinases enabled dsDNA recombineering of Rhodococcus. A CRISPR/Cas9 method was developed for Rhodococcus genome editing for the first time. The engineered Rhodococcus could achieve 50% acrylamide production for 4 batches.