Engineering a Glucosamine-6-phosphate Responsive glmS Ribozyme

Subscriber access provided by Kaohsiung Medical University

Article

Engineering a glucosamine-6-phosphate responsive glmS ribozyme switch enables dynamic control of metabolic flux in Bacillus subtilis for overproduction of N-acetylglucosamine Tengfei Niu, Yanfeng Liu, Jianghua Li, Mattheos A.G. Koffas, Guocheng Du, Hal S. Alper, and Long Liu ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00196 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39 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

ACS Synthetic Biology

1

Engineering a glucosamine-6-phosphate responsive glmS ribozyme switch enables dynamic

2

control of metabolic flux in Bacillus subtilis for overproduction of N-acetylglucosamine

3 4

Tengfei Niu1, 2, Yanfeng Liu1, 2, Jianghua Li1, 2*, Mattheos Koffas3, 4, Guocheng Du1, 2, Hal S. Alper5, 6, Long Liu1, 2*

5 6

1.

7

Wuxi 214122, China

8

2.

Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China

9

3.

Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy NY 12180, USA

10

4.

Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy NY 12180, USA

11

5.

McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712, USA

12

6.

Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, USA

Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University,

13 14

*Corresponding authors: Jianghua Li, Tel.: +86-510-85329031, Fax: +86-510-85918309, E-mail:

15

[email protected]; Long Liu, Tel.: +86-510-85918312, Fax: +86-510-85918309, E-mail:

16

[email protected].

17 18 19 20 21 22 1

ACS Paragon Plus Environment

ACS Synthetic Biology 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

1

Abstract

2

Bacillus subtilis is a typical industrial microorganism and is widely used in industrial biotechnology, particularly

3

for nutraceutical production. There are many studies on the static metabolic engineering of B. subtilis, whereas

4

there are few reports on dynamic metabolic engineering due to the lack of appropriate elements. Here, we

5

established a dynamic reprogramming strategy for reconstructing metabolic networks in B. subtilis, using a

6

typical nutraceutical, N-acetylglucosamine (GlcNAc), as a model product and the glmS (encoding

7

glucosamine-6-phosphate synthase) ribozyme as an engineering element. First, a trp terminator was introduced

8

to effectively release the glmS ribozyme feedback inhibition. Further, we engineered the native

9

glucosamine-6-phosphate (GlcN6P) responsive glmS ribozyme switch to dynamically control the metabolic flux

10

in B. subtilis for overproduction of GlcNAc. With GlcN6P as a ligand, the native sensor glmS ribozyme is

11

integrated at the 5’- of phosphoglucosamine mutase and 6-phosphofructokinase genes to decrease the flux

12

dynamically toward the peptidoglycan synthesis and glycolysis pathway, respectively. The glmS ribozyme

13

mutant M5 (glmS ribozyme cleavage site AG → GG) with decreased ribozyme activity is integrated at the 5’- of

14

glucose-6-phosphate isomerase gene to increase the flux dynamically toward the GlcNAc synthesis pathway.

15

This strategy increased the GlcNAc titer from 9.24 to 18.45 g/L, and the specific GlcNAc productivity from 0.53

16

to 1.21 g GlcNAc/g cell. Since GlcN6P is involved in the biosynthesis of various products, here the developed

17

strategy for multiple target dynamic engineering of metabolic pathways can be generally used in B. subtilis and

18

other industrial microbes for chemical production.

19

Keywords: glmS ribozyme; N-acetylglucosamine; Bacillus subtilis; Dynamic metabolic engineering; GlcN6P

2


Page 2 of 39

Page 3 of 39 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


1

Metabolic engineering is built on a foundation of controlling pathway flux toward a product of

2

interest.

3

through the wholesale overexpression of endogenous and heterologous pathways.1,4,5 However,

4

redirecting metabolic flux through brute-force, unregulated overexpression can inadvertently hamper

5

yield and cell viability. This reality is especially poignant when the desired pathway flux draws from a

6

critical, biomass dependent metabolic intermediate. To address this issue for synthetic systems,

7

several groups have introduced synthetic dynamic control schemes to balance metabolic flux through

8

a branch point in response to metabolite levels.1-3,6-8

9

This work utilizes the host strain Bacillus subtilis, a well-studied, safe, gram-positive bacterium that

10

has a number of unique advantages as an industrially relevant chemicals production host.9,10 To this

11

end, numerous studies have focused on the metabolic engineering of B. subtilis through control of

12

expression strength by promoter engineering,

13

ribosome binding sites.16 The selected product for this study is N-acetylglucosamine (GlcNAc), which

14

has been widely used as food supplements for the management of osteoarthritis.

15

As implied above, most methods for metabolic engineering (esp. in B. subtilis) impart an inherently

16

static control of gene expression, and thus result in undesirable and unbalanced metabolic flux

17

distributions.

18

imbalances.2,20 Moreover, control and balance between cell growth and product synthesis is critical for

19

increasing process metrics of rate, titer, and yield.

20

efficiently increase product synthesis. However, a remaining challenge in the field is designing (or

21

isolating) a responsive sensor to serve as a master regulator in a synthetic, dynamic control

22

scheme.

1-4

In this regard, many endeavors have successfully converted cells into cellular factories

11-13

1,19

13,19

14,15

mRNA stability engineering,

and engineering of

17,18

As a result, titers are often limited due to toxic intermediates and metabolic

3,20-22

The dynamic regulation of the carbon flux can

One common approach is to invoke metabolite-responsive riboswitches to synthetically 3



23-26

Page 4 of 39

23

regulate the expression of critical pathway genes.

24

dynamic range is often self-limiting for several applications. Here, we make use of a unique glmS

25

(encoding glucosamine-6-phosphate synthase; GlmS) ribozyme that resides within noncoding regions

26

of glmS mRNAs in gram-positive bacteria and cleaves its own transcript in response to the intracellular

27

glucosamine 6-phosphate (GlcN6P) concentration (Figure 1A).27,28 In addition, this ribozyme responds

28

linearly to increases in GlcN6P concentrations of about 1000-fold, and the half-life of the precursor

29

RNA is reduced by saturation of the ribozyme with the ligand from approximately 4 h to less than 15

30

s.27 For endogenous systems, feedback regulation of glmS ribozyme sRNA plays an important role in

31

fine-tuning the metabolism of gram-positive bacteria.

32

repression activity can be decreased by site-directed mutagenesis of the self-cleavage site from

33

5’-AG-3’ to 5’-CC-3’ or 5’-AG-3’ to 5’-GG-3’.

34

5’-CC-3’) enables the opposite impact and can stabilize full-length transcripts and ultimately

35

significantly increase reporter gene expression.28

36

In prior work, we constructed a GlcNAc strain using a systems metabolic engineering approach.

37

However, this strain still resulted in significant bottlenecks including low titer and yield as well as a

38

high concentration of the byproduct acetoin. This byproduct in particular demonstrates an imbalance

39

in the synthesis pathway. Here, we use this base strain and engineered the native GlcN6P responsive

40

glmS ribozyme switch to regulate cell growth and GlcNAc synthesis (Figure 1B). To do so, we first

41

engineered glmS ribozyme to release the feedback inhibition (Figure 1B). Next, we utilized the native

42

and mutant glmS ribozymes to both positively and negatively regulate different control targets in

43

these pathways in response to GlcN6P levels. Ultimately, the GlcNAc productivity and yield were

44

increased 2.23-fold by dynamically regulating fluxes to different pathways.

However, the utility of the riboswitch and

27-29

27,28

For synthetic applications, glmS ribozyme

The resulting glmS ribozyme mutant (M9, 5’-AG-3’ to

30-33

34

4


Page 5 of 39 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


45

Results

46

Effect of glmS ribozyme deletion on GlcNAc production

47

The synthesis of GlcNAc precursor GlcN6P was inhibited by the feedback inhibition of GlmS activity by

48

the glmS ribozyme in response to the GlcN6P concentration.27,28 Unexpectedly, deletion of the glmS

49

ribozyme combined with glmS overexpression under the control of the strong constitutive P43

50

promoter (S1-G) decreased GlcNAc production (Figure 2A, B), even though the expression of the glmS

51

gene increased considerably (Figure 2C and Supplementary Figure S1). Therefore, the glmS ribozyme

52

needs to be engineered rationally to increase GlcNAc synthesis, and different strategies were used to

53

delete the glmS ribozyme in order to identify which part of the glmS ribozyme affected GlcNAc

54

production (Figure 2A). We constructed a series of mutants (S2-G, glmS ribozyme 5’ cleavage fragment

55

deletion; S3-G, glmS ribozyme and 5’ cleavage fragment deletion; S4-G, glmS ribozyme expression

56

cassette deletion; S5-G, glmS ribozyme deletion with introduced trp terminator) with glmS gene

57

overexpression under the control of a strong constitutive P43 promoter (Figure 2A). In recombinant

58

strains N6-G, S1-G, S2-G, S3-G, and S4-G, the GlcNAc titers were 9.2, 8.3, 8.6, 8.1, and 8.2 g/L with

59

GlcNAc yields on cell mass of 0.53, 0.53, 0.54, 0.49, and 0.51 g GlcNAc/g dry cell, respectively (Figure

60

2B, D). In S5-G, terminating the expression of the 5’ cleavage fragment by introducing a trp terminator

61

and overexpression of glmS under the control of the strong constitutive promoter P43, gave the highest

62

GlcNAc titer of 12.21 g/L with a GlcNAc yield on cell mass of 0.80 g/g dry cell in shake flasks, which

63

was 32.1% higher than that obtained in control strain N6-G (Figure 2B, D). Compared with N6-G,

64

competition for more carbon from the glycolysis pathway in B. subtilis with the deleted glmS ribozyme

65

strains S1-G, S2-G, S3-G, and S4-G showed a slight decrease in dry cell weight (DCW) (Figure 2D). The

66

DCW (15.26 g/L) of S5-G was the lowest of the mutants, which may be because more flux toward

67

glycolysis was used for GlcNAc synthesis (Figure 2D). 5



Page 6 of 39

68

The relative transcription levels of glmS and the activity of GlmS were analyzed. Compared with N6-G,

69

the glmS mRNA levels of glmS ribozyme deleted strains increased more than three-fold

70

(Supplementary Figure S1). Consequently, the specific GlmS activity in glmS ribozyme deleted strains

71

was higher than that of the control (Figure 2C). The GlcNAc titers and yields of S1-G, S2-G, S3-G, and

72

S4-G have been lower probably because these strategies disrupted the dissociation of the 5’ cleavage

73

fragment RNA and probably inactivated the 5’ cleavage fragment RNA. This would decrease GlcNAc

74

production compared with the strategy of terminating the expression of the 5’ cleavage fragment.

75

Independently dynamic engineering of pathways for improved GlcNAc production

76

We first compartmentalized GlcNAc related biosynthesis networks into 4 sections, namely, the GlcNAc

77

synthesis pathway, pentose phosphate pathway (PPP), glycolysis pathway, and peptidoglycan synthesis

78

pathway (Figure 1B). These four pathways compete for the same precursors, such as

79

glucose-6-phosphate, fructose-6-phosphate and GlcN6P. PFK catalyzes the major rate-limiting step of

80

the glycolysis pathway, and its activity can be used as an indicator of glycolysis in microorganisms.35

81

The glycolysis pathway can be limited by repressing the expression of pfkA. Because GlmM catalyzes

82

the first step of peptidoglycan synthesis from GlcN6P, the flux in the peptidoglycan synthesis pathway

83

can be decreased by downregulating glmM expression. Therefore, we chose pfkA and glmM as targets

84

for downregulating the competitive glycolysis and peptidoglycan synthesis pathways with the glmS

85

ribozyme. Furthermore, glmS ribozyme repression activity can be eliminated by site-directed

86

mutagenesis of the self-cleavage site from 5’-AG-3’ to 5’-CC-3’.

87

mutant M9 enables the opposite impact and can stabilize full-length transcripts and ultimately

88

significantly increase reporter gene expression.

89

pathway, the glmS ribozyme mutant M9 was embedded into the 5’ end of pgi mRNA to increase the

27,28

28

The resulting glmS ribozyme

To increase the flux toward the GlcNAc synthesis

6


Page 7 of 39 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


90

expression of pgi. Because PGI mediates the conversion of glucose-6-phosphate, the starting

91

metabolite of the PPP, into fructose-6-phosphate in the glycolysis pathway, the upregulation of pgi

92

gene expression is expected to switch carbon flux from the PPP to the glycolysis pathway.

93

Compared with N6-G, downregulation of the pfkA gene expression resulted in a 3.2% increase in

94

GlcNAc titer (Supplementary Figure S2A), whereas repressing glmM expression with the glmS

95

ribozyme decreased the GlcNAc titer by 4.3% compared with N6-G (Supplementary Figure S2A).

96

Compared with S5-G, the glycolysis pathway was repressed by the glmS ribozyme in SF-G, the GlcNAc

97

titer was increased from 12.21 to 13.17 g/L (Figure 3A), and GlcNAc yield was increased from 0.80 to

98

1.20 g GlcNAc/g dry cell (Figure 3A, B). Dynamic control of the peptidoglycan synthesis pathway by

99

using the glmS ribozyme in SM-G decreased the GlcNAc titer (Figure 3A) and increased acetoin by 68%

100

(Figure 4A). Cell growth was not affected by the downregulation of the peptidoglycan synthesis

101

pathway due to the increased glycolysis pathway activity (Figure 3B). Compared with S5-G,

102

upregulation of PGI slightly increased the flux toward glycolysis and GlcNAc synthesis according to the

103

increased GlcNAc and acetoin (Figure 3A and Figure 4A). These results indicated that the control of the

104

peptidoglycan synthesis pathway is less effective than that of the glycolysis pathway. Thus, glycolysis

105

was the main type of metabolism, and limiting the metabolic activity of the glycolysis pathway

106

diverted more substrate to GlcNAc synthesis. Moreover, the independently dynamic target has less

107

effect on GlcNAc synthesis due to the carbon flux distributed through uncontrolled pathway.

108

Multiple target dynamic engineering of metabolic pathways for improved GlcNAc production

109

Repressing both glmM and pfkA expression by the glmS ribozyme in FM-G decreased DCW by 10.1%

110

and increased the GlcNAc titer (10.24 g/L) by 10.8% compared with N6-G (Supplementary Figures S2A,

111

B). Compared with S5-G, dynamic downregulation of both the peptidoglycan synthesis and glycolysis 7



112

pathways by the glmS ribozyme in SFM-G substantially decreased the DCW by 23.7% (Figure 3B) and

113

acetoin concentration (20.6 g/L) by 13.4% (Figure 4A), and increased the GlcNAc titer (14.45 g/L)

114

(Figure 3A) by 18.3% and the GlcNAc yield on cell mass by 55% (1.24 g GlcNAc/g dry cell) (Figure 3A, B).

115

These results indicated that simultaneous regulation of both pathways was efficient for increasing

116

GlcNAc synthesis. Although the combinatorial control of pgi and glmM did not increase GlcNAc

117

synthesis, acetoin concentration (36.1 g/L) increased substantially by 51.7% compared with S5-G

118

(Figure 3A and Figure 4A). Compared with S5-G, the combinatorial control of pgi and pfkA increased

119

GlcNAc titer (13.9 g/L) by 13.9% and decreased acetoin concentration (16.6 g/L) by 30.5%. These

120

results demonstrate that two targets combinations dynamic control have more effect on the

121

regulation of carbon flux than that of the independently dynamic target control.

122

On the basis of the simultaneous dynamic control of two pathway fluxes, triple target dynamic

123

engineering was further performed. Compared with SFM-G, upregulating the expression of pgi with

124

the glmS ribozyme mutant M9 increased the GlcNAc titer by 12.5% (16.26 g/L) (Figure 3A) and

125

decreased the DCW by 37.8% (Figure 3B). Unexpectedly, the by-product acetoin was completely

126

eliminated after introducing the glmS ribozyme mutant M9 within pgi mRNA (Figure 4A). Cell growth

127

was decreased by the upregulation of pgi expression, and the GlcNAc yield increased from 1.24 to 2.24

128

g GlcNAc/g dry cell.

129

As for GlcNAc yield on glucose, glmS ribozyme dynamic control of glmM in SM-G decreased GlcNAc

130

yield on glucose from 0.167 to 0.117 g/g glucose (Figure 4B). Similarly, dynamic control glmM and pgi

131

in SMI-G also decreased GlcNAc yield on glucose from 0.167 to 0.131 g/g glucose (Figure 4B). But,

132

dynamic control of pfkA and glmM in SFM-G increased GlcNAc yield on glucose from 0.167 to 0.204

133

g/g glucose (Figure 4B). In addition, triple targets regulation in SFMI-G increased GlcNAc yield on 8


Page 8 of 39

Page 9 of 39 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


134

glucose from 0.167 to 0.394 g/g glucose (Figure 4B). These results indicated that the downregulated

135

pfkA expression diverted more substrate to the GlcNAc synthesis pathway. The regulation of pfkA and

136

glmM by the glmS ribozyme was strengthened by the increased intracellular GlcN6P level due to the

137

upregulated pgi expression (Figure 5A, B, C). These improvements led to an elimination of acetoin

138

production and it seems that this dynamic control system only worked functionally when all of the

139

control targets worked cooperatively in the cell.

140

Obviously, the Pgi activity has a great impact on the carbon flux distribution in pathways. To fine-tune

141

pgi expression for effective GlcNAc synthesis, different elements promoter Phbs, glmS ribozyme and its

142

mutant M5 and RBS2 were adopted (Figure 6A). As compared to SFMI-G, the glmS ribozyme

143

regulating pgi mutants N354-G and N3511-G decreased GlcNAc synthesis (Figure 6B). The GlcNAc titer

144

in mutant N3531-G (the pgi expression was regulated by glmS ribozyme mutant M5) was increased to

145

18.45 g/L, while the mutant N3531-G shows better cell growth and produced more acetoin than that

146

of SFMI-G (Figure 6C, D). Optimal GlcNAc biosynthesis was controlled by feedback regulation system.

147

At low intracellular GlcN6P concentration, the activity of glmS ribozyme mutant M5 decreased,

148

resulting in the increase of Pgi activity and further promotion of GlcN6P accumulation. When there is

149

excessively accumulated GlcN6P, the glmS ribozyme M5 would be activated to repress pgi, resulting in

150

decrease of GlcN6P concentration (Figure 6E). Moderate GlcN6P was then maintained by feedback

151

regulation system and promoted GlcNAc accumulation. Ideally, GlcNAc synthesis would be controlled

152

by the availability of GlcN6P, and glutamine and acetyl-CoA would be produced in no greater than

153

sufficient quantities needed for GlcNAc synthesis. However, there seems to be a contradiction in no

154

influence on cell growth without adequate acetyl-CoA and glutamine. Because GlcNAc synthesis and

155

cell growth compete for the same acetyl-CoA and glutamine. Overall, our results indicated that the 9



156

designed feedback regulation system regulated the cell growth and promoted GlcNAc synthesis.

157

Analysis and verification of function of glmS ribozyme dynamic control systems

158

The relative transcription levels of pfkA and glmM were analyzed further. The pfkA and glmM mRNA

159

levels in SFM-G decreased by 52.7% and 58.1%, respectively, compared with the native expression

160

levels (Supplementary Figure S1). The maximal specific PFK and GlmM activities were repressed to 345

161

and 82 U/mg at 10 h in SFM-G, which were 40% and 48% of the activities without glmS ribozyme

162

regulation, respectively (Figure 5A, B), indicating that the glmS ribozyme approach was effective for

163

repressing specific gene expression in B. subtilis. The intracellular GlcN6P concentrations increased

164

from 3.8 to 10.2 mM at 22 h in SFM-G (Figure 7A). We observed an inverse relationship between

165

intracellular GlcN6P and PFK activity with the regulation of the ribozyme, namely, PFK activity

166

decreased as the GlcN6P concentration increased (Figure 7B). SF-G and SM-G exhibited a similar trend

167

in PFK and GlmM activities (Figure 5A, B). Compared with N6-G, SF-G and SM-G showed low ribozyme

168

activity due to low intracellular GlcN6P concentration (Figure 7A). Furthermore, the PFK and GlmM

169

activities of SFM-G were less than half that of S5-G (Figure 5A, B), suggesting that the ribozyme, when

170

activated by higher intracellular GlcN6P concentration, represses the expression of pfkA and glmM

171

genes (Figure 5A, B, and Figure 7B).

172

SFMI-G exhibited four-fold more pgi mRNA than SFM-G, suggesting that the ribozyme mutant M9

173

stabilizes the pgi mRNA (Supplementary Figure S1). Subsequently, the specific PGI activity in SFMI-G

174

increased to 247 and 230 U/mg at 5 and 22 h, which were 131% and 215% the activity without the

175

glmS ribozyme mutant M9 regulation, respectively (Figure 5C). SFMI-G exhibited lower PFK and GlmM

176

activities than SFM-G (Figure 5A, B). The increase in the intracellular GlcN6P concentration of SFMI-G

177

was even greater than that in SFM-G (Figure 7B). We observed a dynamic circuit configuration in 10


Page 10 of 39

Page 11 of 39 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


178

which the glmS ribozyme activity was activated by intracellular GlcN6P, which increased repression of

179

pfkA and glmM gene expression.

180

To determine if the triple targets regulation increased product yields because of the improved

181

metabolic balance between glycolysis and GlcNAc synthesis pathways, we used both glmS ribozyme

182

and RBS sequence to control the expression of pfkA and compared their effects on GlcNAc synthesis

183

(Supplementary Figure S3A). PA1, PA3, PA5, PA7 and PA8 have varied sequences in the RBS regions of

184

pfkA and cover a range of translation initiation rates from weak to strong. All the cell growth for the

185

RBS variants were consistent with the trend in the PFK activities produced (Supplementary Figure S3B,

186

C). But all the strains having RBS substitution produced considerably less GlcNAc compared to strain

187

SFMI-G (Supplementary Figure S3C). Instead, more acetoin accumulated or less cell growth in these

188

cultures (Supplementary Figure S3C).

189

The pgi expression was fine-tuned by different elements to promote metabolic balance. In

190

comparison with glmS ribozyme mutants, glmS ribozyme lead to a lower intracellular GlcN6P

191

concentrations in the regulation of pgi (Figure 8A). The GlcN6P concentration in N3531-G reached a

192

moderate level of 12.01 mM, which is lower than that of SFMI-G. As a result, the specific PGI activity

193

in N3531-G increased to 179 U/mg at 10 h, which were 87% of that in SFMI-G (Figure 8B). As

194

compared with SFM-G, lower intracellular GlcN6P concentration in N354-G and N3511-G can activate

195

the glmS ribozyme to repress pgi expression, and N354-G and N3511-G represented lower Pgi

196

activities than SFM-G due to the glmS ribozyme feedback inhibition.

197

Discussion

198

Engineering strategies based on biosensors are well established and have been used to obtain efficient

199

microbes for producing industrially important chemicals.

1,2,36

Compared with traditional gene

11



Page 12 of 39

200

knockout, biosensor-dependent engineering strategies have several advantages, such as allowing

201

slight tweaking, higher efficiency, robustness to environmental perturbations, and efficiency in

202

dynamically repressing gene expression, especially for essential genes, which cannot be

203

down-regulated statically by gene knockout approaches.

204

The glmS ribozyme 5’ cleavage fragment RNA can be dissociated by the cleavage reaction from the

205

glmS ribozyme activated by GlcN6P. For deletion of the glmS ribozyme, the glmS ribozyme 5’ cleavage

206

fragment RNA cannot be dissociated without the trp terminator. The trp terminator can restore the

207

dissociation status of the 5’ cleavage fragment RNA to an extent. Our genetic and experimental

208

evidence indicates the GlcNAc production cannot be increased by direct deletion of the glmS ribozyme.

209

The increase requires dissociated mRNA but not long-chain mRNA, because both mRNA with

210

transcriptional termination by a trp terminator and cleaved noncoding RNA are functional. The

211

increase in GlcNAc production is context-dependent because the 5’ cleavage fragment cannot be

212

translated easily in the engineered glmS mRNA without loss of regulation. These observations

213

suggested that the 5’ cleavage fragment pairing with some gene mRNA regulates some processes. We

214

found possible 5’ cleavage fragment RNA pairing with the RBS sequence of ypqE which may influence

215

its protein level (Supplementary Figure S4). Furthermore, previous research showed that the glmS

216

ribozyme mutant M9 (cleavage site AG → CC) strain is incapable of sporulation. This may be because

217

the ribozyme is required to control the glmS gene as well as release a 5’ cleavage fragment. Once

218

liberated from the glmS mRNA, this non-coding RNA might then signal excess GlcN6P concentrations

219

by affecting other cellular processes.27 However, the regulation mechanism of 5’ cleavage fragment

220

RNA remains unknown, and further studies on identifying the mechanism are required to clarify

221

GlcNAc production in B. subtilis.

28

12


Page 13 of 39 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


222

The central carbon pathways constitute the backbone of cell metabolism and provide the energy and

223

reducing power necessary for cell growth.

224

glycolysis and PPP pathways.38 More than half of the carbon flux through the glycolysis pathway is

225

from converting glucose into pyruvate. In this study, acetoin concentrations increased due to carbon

226

flux overflow from the glycolysis pathway. The abnormal acetoin accumulation in N6-G (up to 32 g/L)

227

demonstrated that the glycolysis pathway must be downregulated to increase GlcNAc production.

228

When the glmS ribozyme was used to downregulate both pfkA and glmM, 20 g/L acetoin accumulated

229

(Figure 4A). Furthermore, SFMI-G using the glmS ribozyme mutant M9 prevented overflow

230

metabolism and unwanted acetoin production, suggesting that the central metabolism was balanced

231

with the dynamic regulation (Figure 4A). The decrease in acetoin accumulation and increase in the

232

repression of PFK and GlmM activity was consistent. Notably, we observed a dynamic circuit

233

configuration in which the glmS ribozyme activity was activated by increased intracellular GlcN6P

234

concentrations. These results reveal how carbon flux in central metabolism can be wasted by excess

235

acetoin production.

236

Using natural riboswitches for target gene control is complicated because they typically sense essential

237

metabolites, the concentrations of which may be difficult to control. In this study, GlcN6P functioned

238

as an inducer in a genetically encoded biosensor. The glmS ribozyme cannot be activated by less than

239

5 mM GlcN6P in vivo, and only when the GlcN6P concentration increased to above 10 mM, the glmS

240

ribozyme activity increased. Therefore, the limited dynamic range of the glmS ribozyme demonstrated

241

that it cannot achieve an optimal balance for GlcNAc synthesis at low GlcN6P levels. The glmS

242

ribozyme can be engineered through directed evolution techniques to alter its sensitivity to activation

243

by a lower GlcN6P level. The glmS ribozyme aptamer engineered to respond to different chemical

37

Actively growing bacteria metabolize glucose via the

38

13



244

signals may be screened and selected by SELEX techniques to optimize the metabolic pathway. Thus,

245

the glmS ribozyme can be engineered further to optimize the metabolic pathway for GlcNAc synthesis

246

of B. subtilis.

247

In summary, the naturally existing post-transcriptional sensor, the glmS ribozyme, was rewired as an

248

effective dynamic engineering element to control the GlcNAc biosynthesis of B. subtilis. Multiple

249

targets dynamic control of the gene expression involved in the supply and consumption of GlcN6P

250

resulted in balanced metabolism between the GlcNAc synthesis pathway, peptidoglycan synthesis

251

pathway, glycolysis pathway, and PPP pathway, and greatly increased GlcNAc production without

252

by-product synthesis. This metabolic control allowed the engineered cells to downregulate pathway

253

expression dynamically and increased the metabolic activity of a critical enzyme based on the

254

intracellular level of GlcN6P concentration. In particular, this multiple targets dynamic engineering

255

system only worked functionally when all of the control targets worked cooperatively in the cell, and is

256

meaningful for the metabolic engineering where the desired pathway flux draws from a critical,

257

biomass dependent metabolic intermediate.

258 259

Materials and methods

260

Microorganisms, plasmids and cultivation conditions

261

All strains, plasmids, and primers used in this study are listed in Supplementary Tables 1 and 2. The

262

GlcNAc producer N6 (ΔnagPΔgamPΔgamAΔnagAΔnagBΔldhΔpta::lox72) constructed in our previous

263

work was used as the initial host.31 All microorganisms were grown at 37 °C in Luria-Bertani broth (10

264

g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) or on Luria-Bertani agar plates. The fermentation

265

medium consisted of 6 g/L tryptone, 12 g/L yeast extract, 6 g/L (NH4)2SO4, 12.5 g/L K2HPO4·3H2O, 2.5 14


Page 14 of 39

Page 15 of 39 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


266

g/L KH2PO4, and 100 g/L glucose. Xylose was added to the medium to a final concentration of 5 g/L to

267

induce expression of genes under the control of the xylose-inducible PxylA promoter. For selection, 20

268

mg/L kanamycin and 20 mg/L zeocin were used. B. subtilis transformation was performed via

269

electrotransformation.

270

Gene knockout and strain construction.

271

The marker-free knockout approach was used to delete and integrate genes successively. The front

272

and back sequences (approximately 700 bp) flanking the deletion target were amplified by the PCR

273

with corresponding primers (Table 2). These two fragments and the lox71-zeo-lox66 cassettes,

274

amplified from p7Z6, were fused via PCR into the deletion cassette. The deletion cassette was used to

275

transform

276

temperature-sensitive plasmid pTSC was introduced into a zeo clone to remove the resistance marker

277

cassette by the specific recombination of lox71 and lox66. The pTSC vector was cured by incubating at

278

50 °C to obtain a strain without the selected marker and plasmid.

279

To eliminate the feedback inhibition guided by the glmS ribozyme, the glmS ribozyme

280

GlcN6P-responsive portion (Figure 1A) was replaced by the P43 promoter from N6, yielding the

281

recombinant strain, S1. The glmS ribozyme 5’ cleavage fragment, intact glmS ribozyme, and glmS

282

ribozyme cassette were replaced by the P43 promoter sequence from N6, yielding S2, S3, and S4,

283

respectively. S5 was the trp terminator-P43 promoter cassette substitution of the glmS ribozyme

284

GlcN6P-responsive portion from N6 in the genome. Plasmid pP43-GNA1 was transformed into S1, S2,

285

S3, S4, and S5, yielding S1-G, S2-G, S3-G, S4-G, and S5-G, respectively.

286

To downregulate the glycolysis and peptidoglycan pathways to increase GlcNAc production, the glmS

287

ribozyme was embedded in the 5’ untranslated regions of the pfkA mRNA from N6 and S5, yielding the

39

40

B.

subtilis

competent

cells,

and

zeor

transformants

were

selected.

The

r

15



Page 16 of 39

288

mutants F and SF, and was embedded within the 5’ untranslated regions of the glmM mRNA from N6

289

and S5, yielding the mutants M and SM, respectively. Finally, FM and SFM were obtained by

290

embedding the glmS ribozyme in the upstream of the glmM mRNA from F and SF. Plasmid pP43-GNA1

291

was transformed into F, M, FM, SF, SM, and SFM, yielding F-G, M-G, FM-G, SF-G, SM-G, and SFM-G,

292

respectively.

293

To upregulate the key metabolic node pgi expression for promoting flux toward the GlcNAc synthesis

294

pathway, the glmS ribozyme mutant M9 was integrated into the 5’-flanking region of pgi mRNA from

295

S5, SF, SM, SFM, yielding the recombinant strain SI, SFI, SMI and SFMI, respectively. Plasmid

296

pP43-GNA1 was transformed into SI, SFI, SMI and SFMI, yielding SI-G, SFI-G, SMI-G and SFMI-G,

297

respectively.

298

An RBS mutant library of PFK was designed using the RBS calculator. The primers were designed to

299

match the target sequence to construct deletion cassette. The deletion cassette was used to transform

300

SFMI competent cells, yielding the recombinant strain PA1, PA3, PA5, PA7 and PA8. Plasmid

301

pP43-GNA1 was transformed into PA1, PA3, PA5, PA7 and PA8, yielding PA1-G, PA3-G, PA5-G, PA7-G

302

and PA8-G, respectively.

303

To regulate the dynamic expression of pgi for promoting flux towards the GlcNAc synthesis pathway,

304

different elements including promoter Phbs,

305

AG→GG), and RBS2 (5’-ATATTTAAGAGGAGGAATTAA-3’) were used to tune the pgi expression from

306

SFM, yielding the recombinant strain N351, N352, N353, N354, N3511, and N3531, respectively.

307

Plasmid pP43-GNA1 was transformed into N351, N352, N353, N354, N3511, and N3531, yielding

308

N351-G, N352-G, N353-G, N354-G, N3511-G, and N3531-G, respectively.

309

Analytical methods

41

42

the glmS ribozyme and its mutants M5 (cleavage cite

16


Page 17 of 39 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


310

The concentrations of GlcNAc, GlcN6P, and acetoin were measured by high-performance liquid

311

chromatography (HPLC) (Agilent 1260, Bio-Rad, Hercules, CA; HPX-87H column) and a refractive index

312

detector using 5 mM H2SO4 as the mobile phase at a flow rate of 0.6 mL/min and 35 °C.

313

Glucose concentration in the supernatant was measured with a glucose-glutamate analyzer (SBA-40C,

314

Biology Institute of Shandong Academy of Sciences, Jinan, China). The DCW per liter was calculated

315

according to the experimentally determined formula DCW (g/L) = 0.35 × OD600.

316

concentration of GlcN6P was calculated with 1 mL of a B. subtilis culture with an OD600 of 1 and a total

317

cell volume of 0.56 μL.

318

For intracellular GlcN6P assays, cells were harvested at given times and disrupted by freeze-thaw

319

cycles and sonication. After centrifugation at 10,000 g for 10 min, the supernatant was used for HPLC

320

analysis. The supernatant was also used for GlmS, PFK, GlmM, and PGI activity assays according to a

321

previously reported protocol.

322

formed per milligrams of protein in 1 min under the assay conditions.

323

For key gene transcription assays, cells were harvested during fermentation via centrifugation at 1000

324

g for 2 min. Total RNA was extracted from the harvested cells using a Simply P Total RNA Extraction kit

325

(BioFlux, China). The DNA in the mRNA extract samples was removed by DNase I. Purified mRNA was

326

transcribed into cDNA by using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, China) with a

327

random hexamer primer. The cDNA was used for real-time PCR with a RealMasterMix (SYBR Green) kit

328

(Tiangen, China), and then relative gene expression analysis was performed.

31

35,38,43,44

The intracellular

One unit of enzyme was defined as the picomoles of product

329 330

Acknowledgements

331

This work was financially supported by the National Natural Science Foundation of China (31622001, 17



332

31671845, 21676119 and 31600068), the Natural Science Foundation of Jiangsu Province

333

(BK20160176), and the 111 Project (No. 111-2-06).

334

335

Author contributions

336

T.N., Y.L., J.L., G.D., H.A. and L.L. designed the experiments. T.N. performed the experiments. T.N., G.D.,

337

M.K., H.A. and L.L. conceived the project, analyzed the data, and wrote the paper.

338

339

Competing financial interests

340

The authors declare that they have no competing financial interests.

341

18


Page 18 of 39

Page 19 of 39 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


342

Table 1. Strains and plasmids used in this study. Names

Characteristics

Reference

strains N6

31

B. subtilis 168 derivate, ΔnagPΔgamPΔgamAΔnagAΔnagBΔldhΔpta::lox72

different glmS ribozyme portions deletion strains S1

N6 derivate, substitution of the glmS ribozyme

This work

GlcN6P-responsive portion by P43 promoter S2

N6 derivate, substitution of glmS ribozyme 5’ cleavage fragment

This work

by P43 promoter S3

N6 derivate, substitution of the intact glmS ribozyme by P43

This work

promoter S4

N6 derivate, substitution of glmS ribozyme cassette by P43

This work

promoter S5

N6 derivate, substitution of glmS ribozyme GlcN6P-responsive

This work

portion by trp terminator-P43 promoter cassette glmS ribozyme and mutant control strains SF

S5 derivate, embedding glmS ribozyme into 5’ end of pfkA mRNA

This work

SM

S5 derivate, embedding glmS ribozyme into 5’ end of glmM

This work

mRNA SI

S5 derivate, embedding glmS ribozyme mutant M9 (cleavage site

This work

AG→CC) into 5’ end of pgi mRNA SFM

SF derivate, embedding glmS ribozyme into 5’ end of glmM

This work

mRNA SFI

SF derivate, embedding glmS ribozyme mutant M9 (cleavage site

This work

AG→CC) into 5’ end of pgi mRNA SMI

SM derivate, embedding glmS ribozyme mutant M9 (cleavage

This work

site AG→CC) into 5’ end of pgi mRNA SFMI

SFM derivate, embedding glmS ribozyme mutant M9 (cleavage

This work

site AG→CC) into 5’ end of pgi mRNA different engineering elements optimize pgi expression strains N351

SFM derivate, embedding glmS ribozyme mutant (cleavage site

This work

AG→GG) into 5’ end of pgi mRNA N352

SFM derivate, substitution of glmS promoter and RBS1 sequence by hbs promoter and RBS2 sequence 19


This work


N353

SFM derivate, substitution of glmS promoter and RBS1 sequence

Page 20 of 39

This work

by Phbs-glmS ribozyme mutant (cleavage site AG→GG)-RBS2 construct N354

SFM derivate, substitution of glmS promoter and RBS1 sequence

This work

by Phbs-glmS ribozyme-RBS2 construct N3511

SFM derivate, embedding glmS ribozyme into 5’ end of pgi mRNA

This work

N3531

SFM derivate, substitution of glmS promoter by Phbs-glmS

This work

ribozyme mutant (cleavage site AG→GG) construct GNA1 expression strains N6-G

N6 derivate, overexpression of S. cerevisiae 168 GNA1 gene

31

S1-G

S1 derivate, overexpression of S. cerevisiae 168 GNA1 gene

This work

S2-G


This work

S3-G


This work

S4-G


This work

S5-G


This work

SF-G

SF derivate, overexpression of S. cerevisiae 168 GNA1 gene

This work

SM-G

SM derivate, overexpression of S. cerevisiae 168 GNA1 gene

This work

SI-G

SI derivate, overexpression of S. cerevisiae 168 GNA1 gene

This work

SFM-G

SFM derivate, overexpression of S. cerevisiae 168 GNA1 gene

This work

SFI-G

SFI derivate, overexpression of S. cerevisiae 168 GNA1 gene

This work

SMI-G

SMI derivate, overexpression of S. cerevisiae 168 GNA1 gene

This work

SFMI-G

SFMI derivate, overexpression of S. cerevisiae 168 GNA1 gene

This work

SFM1-G

SFM1 derivate, overexpression of S. cerevisiae 168 GNA1 gene

This work

SFM2-G


This work

SFM3-G


This work

N351-G


This work

N352-G


This work

N353-G


This work

N354-G


This work

N3511-G


This work

N3531-G


This work

Plasmids p7Z6

pMD18-T containing lox71-zeo-lox66 cassette

40

pTSC

EmrAmpr; temperature sensitive in B. subtilis

40

pP43-GNA1

pP43NMK derivate with GNA1 cloned

30

20


Page 21 of 39 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


1

Table 2. Primers used in this study. Primer

Sequence (5’-3’)

glmS1-L-F

GCAGATGTTTCTACAATGGGGAC

glmS1-L-R

CTGTTTCCTGTGTGAAATTGTTATCCGCTCGGTAAAGCAATAATCCCGTC

glmS1-Z-F

GACGGGATTATTGCTTTACCGAGCGGATAACAATTTCACACAGGAAACAG

glmS1-Z-R

GCGAAAACATACCACCTATCAGCCAGGGTTTTCCCAGTCACGAC

glmS1-P43-F

GTCGTGACTGGGAAAACCCTGGCTGATAGGTGGTATGTTTTCGC

glmS1-P43-R

CGTCCCCTCCTACATGTTTTTATAATGGTACCGCTATCAC

glmS1-R-F

GTGATAGCGGTACCATTATAAAAACATGTAGGAGGGGACG

glmS1-R-R

CATCAGATGCTACGACGTTG

glmS2-L-F

AAATCAGAGGGCCGTTTAAAGGATG

glmS2-L-R

GCTATACGAACGGTAGAATCTCGACAAGCTTGATTTTACAACATGTGGC

glmS2-Z-F

GCCACATGTTGTAAAATCAAGCTTGTCGAGATTCTACCGTTCGTATAGC

glmS2-Z-R

GCGAAAACATACCACCTATCACTACCGTTCGTATAATGTATGC

glmS2-P43-F


glmS2-P43-R

TTTTTCCGGGCGCTTAGTTCGGGCGGGTATAATGGTACCGCTATCAC

glmS2-R-F

GTGATAGCGGTACCATTATACCCGCCCGAACTAAGCGCCCGGAAAAA

glmS2-R-R

GGAACGCTTCTTCTGTCTCAAGTCC

glmS3-L-F

AAATCAGAGGGCCGTTTAAAGGATG

glmS3-L-R

GCTATACGAACGGTAGAATCTCGACAAGCTTGATTTTACAACATGTGGC

glmS3-Z-F

GCCACATGTTGTAAAATCAAGCTTGTCGAGATTCTACCGTTCGTATAGC

glmS3-Z-R


glmS3-P43-F


glmS3-P43-R

CGTCCCCTCCTACATGTTTTTATAATGGTACCGCTATCAC

glmS3-R-F

GTGATAGCGGTACCATTATAAAAACATGTAGGAGGGGACG

glmS3-R-R

GGAACGCTTCTTCTGTCTCAAGTCC

glmS4-L-F

TCCCCGAACGGATTAAACAT

glmS4-L-R

TATACGAACGGTAGAATCTCTAGACTTCGTTACTCTAATCCC

glmS4-Z-F

GGGATTAGAGTAACGAAGTCTAGAGATTCTACCGTTCGTATA

glmS4-Z-R


glmS4-P43-F

GCATACATTATACGAACGGTAGTGATAGGTGGTATGTTTTCGC

glmS4-P43-R

CCTAAGATTGTAAAAGGAGACGCCGCTATCACTTTATATTTTACATAATCGC

glmS4-R-F

GCGATTATGTAAAATATAAAGTGATAGCGGCGTCTCCTTTTACAATCTTAGG

glmS4-R-R

CTCCAAGACCTACTAATAGAG

glmS5-L-R (ter)

GTAGAATCTCAAAAAAGCCCGCTCATTAGGCGGGCTGCCTTTTTCCGGGCGCTTAGTT 21



glmS5-Z-F (ter)

GGAAAAAGGCAGCCCGCCTAATGAGCGGGCTTTTTTGAGATTCTACCGTTCGTATA

pfkA-SR-L-F

CGAACACCTGTTTACCGACTT

pfkA-SR-L-R

GCTATACGAACGGTAGAATCTCCCTCAGCAACATATATGATTAAACATAACA

pfkA-SR-Z-F

TGTTATGTTTAATCATATATGTTGCTGAGGGAGATTCTACCGTTCGTATAGC

pfkA-SR-Z-R

CCTATTGAGAAAATAAGAACAAGACAAGCTCTACCGTTCGTATAATGTATGC

pfkA-SR-F

GCATACATTATACGAACGGTAGAGCTTGTCTTGTTCTTATTTTCTCAATAGG

pfkA-SR-R

TTCTCCATTCACCTCAGCAACAAGATTGTAAAAGGAGACGAAGAAAGTCAAA

pfkA-SR-R-F

TTTGACTTTCTTCGTCTCCTTTTACAATCTTGTTGCTGAGGTGAATGGAGAA

pfkA-SR-R-R

AATACTGTGCTTCTTGCCGCGTT

glmM-SR-L-F

AAATTGAACGGACAGGAAGCC

glmM-SR-L-R

GCTATACGAACGGTAGAATCTCTTATTCCGATGAGGATTGTG

glmM-SR-Z-F

CACAATCCTCATCGGAATAAGAGATTCTACCGTTCGTATAGC

glmM-SR-Z-R

CCTATTGAGAAAATAAGAACAAGACAAGCTCTACCGTTCGTATAATGTATGC

glmM-SR-F

GCATACATTATACGAACGGTAGAGCTTGTCTTGTTCTTATTTTCTCAATAGG

glmM-SR-R

CTTGCCCATTTTATAATCGCTCCTTTTGATTGTAAAAGGAGACGAAG

glmM-SR-R-F

CTTCGTCTCCTTTTACAATCAAAAGGAGCGATTATAAAATGGGCAAG

glmM-SR-R-R

CAAGACCGAGATCCGCGTTTTT

pgi-SRM-L-F

GGTTGACATGATGAGCCACGTATTC

pgi-SRM-L-R

GCTATACGAACGGTAGAATCTCCCATAACGGTATAATGTTTTCATCTTTCACTTTAT

pgi-SRM-Z-F

ATAAAGTGAAAGATGAAAACATTATACCGTTATGGGAGATTCTACCGTTCGTATAGC

pgi-SRM-Z-R

CGGGCGGGATAATTATAGGTAAAGCCTACCGTTCGTATAATGTATGC

pgi-SRM-F

GCATACATTATACGAACGGTAGGCTTTACCTATAATTATCCCGCCCG

pgi-SRM-R

GTCATTGCTTGTCCCTCCATAACGGACTTTCAATCGTCCCCTCCTACATG

pgi-SRM-R-F

CATGTAGGAGGGGACGATTGAAAGTCCGTTATGGAGGGACAAGCAATGAC

pgi-SRM-R-R

CTGACAGCAATCGGCAAGAGACCTA

pgi-SRM5-Z-R

CGGGCGCCATAATTATAGGTAAAGCCTACCGTTCGTATAATGTATGC

pgi-SRM5-F

GCATACATTATACGAACGGTAGGCTTTACCTATAATTATGGCGCCCG

pgi-hbs-L-F

GCTGGTACTATTGCTTTAAACGG

pgi-hbs-L-R

GCTATACGAACGGTAGAATCTCCTGTGAAATCAAGTCAAGAAGAAAGGGC

pgi-hbs-Z-F

GCCCTTTCTTCTTGACTTGATTTCACAGGAGATTCTACCGTTCGTATAGC

pgi-hbs-Z-R

TTTTTCCTTTTTTCATCCTATTCCTTGATCCTCTACCGTTCGTATAATGTATGC

pgi-hbs-F

GCATACATTATACGAACGGTAGAGGATCAAGGAATAGGATGAAAAAAGGAAAAA

pgi-hbs-R

GAGTAGTCAAAGCGTACATGCGTCATTTAATTCCTCCTCTTAAATATAATCCCAAAAGGAT ACATTCAGTTCGTTTATTA

pgi-hbs-R-F

TTTGGGATTATATTTAAGAGGAGGAATTAAATGACGCATGTACGCTTTGACTACTC 22


Page 22 of 39

Page 23 of 39 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


pgi-hbs-R-R

GCCGCGCTCTTTATCAGTTG

pgi-hbs-R(SRM5)

CGGGCGCCATAATTATAGGTAAAGCCCCAAAAGGATACATTCAGTTCGTTTATTA

pgi-hbs-SRM5-F

TAATAAACGAACTGAATGTATCCTTTTGGGGCTTTACCTATAATTATGGCGCCCG

pgi-hbs-SR/M5-R

GAGTAGTCAAAGCGTACATGCGTCATTTAATTCCTCCTCTTAAATATAATACTTTCAATCGT CCCCTCCTACATG

pgi-hbs-SR/M5-R-F

TGAAAGTATTATATTTAAGAGGAGGAATTAAATGACGCATGTACGCTTTGACTACTC

pgi-hbs-R(SR)

CGGGCGCTATAATTATAGGTAAAGCCCCAAAAGGATACATTCAGTTCGTTTATTA

pgi-hbs-SR-F

TAATAAACGAACTGAATGTATCCTTTTGGGGCTTTACCTATAATTATAGCGCCCG

pgi-SRM5-Z-R

CGGGCGCTATAATTATAGGTAAAGCCTACCGTTCGTATAATGTATGC

pgi-SRM5-F

GCATACATTATACGAACGGTAGGCTTTACCTATAATTATAGCGCCCG

pgi-hbs-

GAGTAGTCAAAGCGTACATGCGTCATTGCTTGTCCCTCCATAACGGTATAAACTTTCAATC

SR/M5-R31

GTCCCCTCCTACATG

pgi-hbs-SR/M5-R-F

TTATACCGTTATGGAGGGACAAGCAATGACGCATGTACGCTTTGACTACTC

21

1

Underlined letters represent homologous sequences for fusion PCR. The cleavage sites are indicated in

2

boldface.

3

23



Page 24 of 39

1

Figure legends

2

Figure 1. Mechanism of glmS mRNA destabilization by the glmS ribozyme and GlcNAc synthesis

3

pathway. (A) The glmS mRNA comprised a glmS ribozyme domain (green) and the ORF (yellow)

4

encoding the protein Glucosamine-6-phosphate synthetase (GlmS). The enzyme catalyzes conversion

5

of fructose-6-phosphate and glutamine into glutamate and glucosamine-6-phosphate (GlcN6P). When

6

GlcN6P accumulates cytoplasmically, it binds to the glmS ribozyme domain, activating a latent

7

self-cleavage activity. This releases the leader sequence, exposing a new 5’-OH terminus in the cleaved

8

mRNA. RNase J1 specifically recognizes the 5’-OH, and degrades the glmS mRNA. The degradation of

9

glmS mRNA resulted in decrease in GlmS activity to lower GlcN6P synthesis. (B) Metabolic pathway

10

of GlcNAc and GlcN6P in B. subtilis. F-6-P, fructose-6-phosphate; G-6-P, glucose-6-phosphate; GlcN6P,

11

glucosamine-6-phosphate;

12

N-acetylglucosamine-1-phosphate; GlcN1P, glucosamine-1-phosphate; GlcNAc, N-acetylglucosamine;

13

PYR, pyruvate; PEP, phosphoenolpyruvic acid; FBP, fructose-1,6-bis-phosphate; GlmS, glucosamine

14

synthase;

15

6-phosphofructokinase; GlmM, phosphoglucosamine mutase.

28

GNA1,

GlcN6P

GlcNAc6P,

N-acetylglucosamine-6-phosphate;

N-acetyltransferase;

PGI,

glucose-6-phosphate

GlcNAc1P,

isomerase;

PFK,

16 17

Figure 2. Different strategies of glmS ribozyme deletion and influence on GlcNAc synthesis. (A) Initial

18

host (N6-G) and different glmS ribozyme deletion strategies. (B) GlcNAc concentration. (C) Influence of

19

glmS ribozyme deletion strategies on intracellular GlmS activities. (D) Cell growth profiles of B. subtilis

20

recombinants N6-G (initial host, pink squares), S1-G (glmS ribozyme deletion, yellow circles), S2-G

21

(glmS ribozyme 5’ cleavage fragment deletion, blue triangles), S3-G (glmS ribozyme and 5’ cleavage

22

fragment deletion, gray asteroids), S4-G (glmS ribozyme expression cassette deletion, green 24


Page 25 of 39 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


1

diamonds), S5-G (glmS ribozyme deletion with introduced trp terminator, purple inverted triangles).

2

Each point represents the average of three independent experiments and error bars represent

3

standard deviation.

4 5

Figure 3. The influence of glmS ribozyme and its mutant multiple targets regulation on GlcNAc

6

synthesis and cell growth. (A) GlcNAc concentration, (B) cell growth profiles of N6-G (control, black

7

squares), S5-G (glmS ribozyme deletion with introduced trp terminator, orange circles), SI-G (glmS

8

ribozyme mutant M9 regulating pgi mRNA, blue open triangles), SF-G (glmS ribozyme regulating pfkA

9

mRNA, purple open inverted triangles), SM-G (glmS ribozyme regulating glmM mRNA, red open

10

diamonds), SFI-G (glmS ribozyme regulating pfkA mRNA and glmS ribozyme mutant M9 regulating pgi

11

mRNA, blue inclined triangles), SMI-G (glmS ribozyme regulating glmM mRNA and glmS ribozyme

12

mutant M9 regulating pgi mRNA, red inclined triangles), SFM-G (glmS ribozyme regulating pfkA and

13

glmM mRNA, green hexagons), and SFMI-G (glmS ribozyme regulating pfkA and glmM mRNA and glmS

14

ribozyme mutant M9 regulating pgi mRNA, brown asteroids) by flask cultures. Values and error bars

15

represent the mean and s.d. of triplicate experiments.

16 17

Figure 4. The influence of glmS ribozyme and its mutant multiple targets regulation on acetoin

18

synthesis and yield. (A) by-product acetoin titer profiles of N6-G (control, black squares), S5-G (glmS

19

ribozyme deletion with introduced trp terminator, orange circles), SI-G (glmS ribozyme mutant M9

20

regulating pgi mRNA, blue open triangles), SF-G (glmS ribozyme regulating pfkA mRNA, purple open

21

inverted triangles), SM-G (glmS ribozyme regulating glmM mRNA, red open diamonds), SFI-G (glmS

22

ribozyme regulating pfkA mRNA and glmS ribozyme mutant M9 regulating pgi mRNA, blue inclined 25



1

triangles), SMI-G (glmS ribozyme regulating glmM mRNA and glmS ribozyme mutant M9 regulating pgi

2

mRNA, red inclined triangles), SFM-G (glmS ribozyme regulating pfkA and glmM mRNA, green

3

hexagons), and SFMI-G (glmS ribozyme regulating pfkA and glmM mRNA and glmS ribozyme mutant

4

M9 regulating pgi mRNA, brown asteroids), (B) GlcNAc yield on glucose. Values and error bars

5

represent the mean and s.d. of triplicate experiments.

6 7

Figure 5. Activity profiles in crude lysates indicate the dynamic regulation. (A) PFK activities under

8

dynamic regulation of glmS ribozyme at given times; (B) GlmM activities under dynamic regulation of

9

glmS ribozyme at given times; (C) PGI activities under dynamic regulation of glmS ribozyme at given

10

times. Values and error bars represent the mean and s.d. of triplicate experiments.

11 12

Figure 6. Dynamic control pgi expression regulates cell growth and GlcNAc synthesis. (A) Different

13

elements were used to control pgi expression. Wild-type glmS ribozyme (cleavage cite AG) and

14

decreased ribozyme activity (cleavage cite AG→GG and AG→CC) were used to control pgi expression.

15

(B) GlcNAc concentrations, (C) cell growth, (D) acetoin concentrations profiles of SFM-G (grey bars),

16

SFMI-G (pink bars), N351-G (cyan bars), N352-G (yellow bars), N353-G (green bars), N354-G (orange

17

bars), N3511-G (purple bars), and N3531-G (red bars) by flask cultures. Values and error bars represent

18

the mean and s.d. of triplicate experiments. (E) GlcNAc biosynthesis pathway was controlled by

19

feedback regulation system. This system contains the sensors glmS ribozyme and its mutant, the

20

ligand GlcN6P and sensor-regulated targets (pfkA, glmM, pgi). When there is inadequate GlcN6P

21

accumulation, enhanced expression of Pgi and repressed expression of pfkA, glmM by glmS ribozyme

22

increase GlcN6P accumulation. When GlcN6P accumulates to a certain extent, it activates glmS 26


Page 26 of 39

Page 27 of 39 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


1

ribozyme mutant to repress pgi and GlcN6P accumulation reduces. Moderate level of GlcN6P was then

2

maintained by feedback regulation system and promotion of GlcNAc accumulation was achieved.

3 4

Figure 7. Functionality of glmS ribozyme dynamic regulation in response to intracellular GlcN6P

5

concentrations in multiple culture media. (A) Intracellular GlcN6P concentrations profiles at a given

6

time. (B) PFK activity plotted against intracellular GlcN6P at a given time (10 h post inoculation).

7

Values and error bars represent the mean and s.d. of triplicate experiments.

8 9

Figure 8. Functionality of glmS ribozyme and its mutants dynamic regulation on pgi expression in

10

response to intracellular GlcN6P concentrations in multiple culture media. (A) Intracellular GlcN6P

11

concentrations profiles at a given time (10 h post inoculation). (B) Pgi activities under dynamic

12

regulation of different elements at given times. Values and error bars represent the mean and s.d. of

13

triplicate experiments.

14

27



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

References 1.

Zhang, F., Carothers, J. M., Keasling, J. D. (2012) Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat. Biotechnol. 30, 354-359.

2.

Dahl, R. H., Zhang, F., Alonso-Gutierrez, J., Baidoo, E., Batth, T. S., Redding-Johanson, A. M., Petzold, C. J., Mukhopadhyay, A., Lee, T. S., Adams, P. D., Keasling, J. D. (2013) Engineering dynamic pathway regulation using stress-response promoters. Nat. Biotechnol. 31, 1039-1046.

3.

Gupta, A., Reizman, I. M., Reisch, C. R., Prather, K. L. (2017) Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit. Nat. Biotechnol. 35, 273-279.

4.

Chen, X., Gao, C., Guo, L., Hu, G., Luo, Q., Liu, J., Nielsen, J., Chen, J., Liu, L. (2018) DCEO Biotechnology: Tools To Design, Construct, Evaluate, and Optimize the Metabolic Pathway for Biosynthesis of Chemicals. Chem. Rev. 118, 4-72.

5.

Pitera, D. J., Paddon, C. J., Newman, J. D., Keasling, J. D. (2007) Balancing a heterologous mevalonate pathway for improved isoprenoid production in Escherichia coli. Metab. Eng. 9, 193-207.

6.

Xu, P., Gu, Q., Wang, W., Wong, L., Bower, A. G. W., Collins, C. H., Koffas, M. A. G. (2013) Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat. Commun. 4.

7.

Venayak, N., Anesiadis, N., Cluett, W. R., Mahadevan, R. (2015) Engineering metabolism through dynamic control. Curr. Opin. Biotechnol. 34, 142-152.

8.

Brockman, I. M., Prather, K. L. (2015) Dynamic knockdown of E. coli central metabolism for redirecting fluxes of primary metabolites. Metab. Eng. 28, 104-113.

9.

Schumann, W. (2007) Production of recombinant proteins in Bacillus subtilis. Adv. Appl.

10.

Shi, S., Chen, T., Zhang, Z., Chen, X., Zhao, X. (2009) Transcriptome analysis guided metabolic

Microbiol. 62, 137-189. engineering of Bacillus subtilis for riboflavin production. Metab. Eng. 11, 243-252. 11.

Hammer, K., Mijakovic, I., Jensen, P. R. (2006) Synthetic promoter libraries-tuning of gene expression. Trends Biotechnol. 24, 53-55.

12.

Miksch, G., Bettenworth, F., Friehs, K., Flaschel, E., Saalbach, A., Twellmann, T., Nattkemper, T. W. (2005) Libraries of synthetic stationary-phase and stress promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli. J. Biotechnol. 120, 25-37.

13.

Cox, R. S., 3rd, Surette, M. G., Elowitz, M. B. (2007) Programming gene expression with combinatorial promoters. Mol. Syst. Biol. 3, 145.

14.

Smolke, C. D., Martin, V. J., Keasling, J. D. (2001) Controlling the metabolic flux through the carotenoid pathway using directed mRNA processing and stabilization. Metab. Eng. 3, 313-321.

15.

Pfleger, B. F., Pitera, D. J., Smolke, C. D., Keasling, J. D. (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat. Biotechnol. 24, 1027-1032.

16.

Salis, H. M., Mirsky, E. A., Voigt, C. A. (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946-950. 28


Page 28 of 39

Page 29 of 39 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


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

17.

Chen, J. K., Shen, C. R., Liu, C. L. (2010) N-acetylglucosamine: production and applications.

18.

Kubomura, D., Ueno, T., Yamada, M., Nagaoka, I. (2017) Evaluation of the chondroprotective

Mar. Drugs. 8, 2493-2516. action of N-acetylglucosamine in a rat experimental osteoarthritis model. Exp. Ther. Med. 14, 3137-3144. 19.

Cress, B. F., Trantas, E. A., Ververidis, F., Linhardt, R. J., Koffas, M. A. (2015) Sensitive cells: enabling tools for static and dynamic control of microbial metabolic pathways. Curr. Opin. Biotechnol. 36, 205-214.

20.

Liu, Y., Li, J., Du, G., Chen, J., Liu, L. (2017) Metabolic engineering of Bacillus subtilis fueled by systems biology: Recent advances and future directions. Biotechnol. Adv. 35, 20-30.

21.

Solomon, K. V., Sanders, T. M., Prather, K. L. (2012) A dynamic metabolite valve for the control of central carbon metabolism. Metab. Eng. 14, 661-671.

22.

Paradise, E. M., Kirby, J., Chan, R., Keasling, J. D. (2008) Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by down-regulating squalene synthase. Biotechnol. Bioeng. 100, 371-378.

23.

Wittmann, A., Suess, B. (2012) Engineered riboswitches: Expanding researchers’ toolbox with

24.

Wachsmuth, M., Findeiss, S., Weissheimer, N., Stadler, P. F., Morl, M. (2013) De novo design

synthetic RNA regulators. FEBS Lett. 586, 2076-2083. of a synthetic riboswitch that regulates transcription termination. Nucleic Acids Res. 41, 2541-2551. 25.

Liu, D., Evans, T., Zhang, F. (2015) Applications and advances of metabolite biosensors for metabolic engineering. Metab. Eng. 31, 35-43.

26.

McKeague, M., Wong, R. S., Smolke, C. D. (2016) Opportunities in the design and application of RNA for gene expression control. Nucleic Acids Res. 44, 2987-2999.

27.

Winkler, W. C., Nahvi, A., Roth, A., Collins, J. A., Breaker, R. R. (2004) Control of gene

28.

Collins, J. A., Irnov, I., Baker, S., Winkler, W. C. (2007) Mechanism of mRNA destabilization by

expression by a natural metabolite-responsive ribozyme. Nature. 428, 281-286. the glmS ribozyme. Genes Dev. 21, 3356-3368. 29.

Bingaman, J. L., Zhang, S., Stevens, D. R., Yennawar, N. H., Hammes-Schiffer, S., Bevilacqua, P. C. (2017) The GlcN6P cofactor plays multiple catalytic roles in the glmS ribozyme. Nat. Chem. Biol. 13, 439-445.

30.

Liu, Y., Liu, L., Shin, H. D., Chen, R. R., Li, J., Du, G., Chen, J. (2013) Pathway engineering of Bacillus subtilis for microbial production of N-acetylglucosamine. Metab. Eng. 19, 107-115.

31.

Liu, Y., Zhu, Y., Li, J., Shin, H.-d., Chen, R. R., Du, G., Liu, L., Chen, J. (2014) Modular pathway engineering of Bacillus subtilis for improved N-acetylglucosamine production. Metab. Eng. 23, 42-52.

32.

Liu, Y., Zhu, Y., Ma, W., Shin, H. D., Li, J., Liu, L., Du, G., Chen, J. (2014) Spatial modulation of key pathway enzymes by DNA-guided scaffold system and respiration chain engineering for improved N-acetylglucosamine production by Bacillus subtilis. Metab. Eng. 24, 61-69.

33.

Liu, Y., Link, H., Liu, L., Du, G., Chen, J., Sauer, U. (2016) A dynamic pathway analysis approach reveals a limiting futile cycle in N-acetylglucosamine overproducing Bacillus subtilis. Nat. Commun. 7, 11933.

34.

Gu, Y., Deng, J., Liu, Y., Li, J., Shin, H. D., Du, G., Chen, J., Liu, L. (2017) Rewiring the glucose transportation and central metabolic pathways for overproduction of N-acetylglucosamine in 29



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

Bacillus subtilis. Biotechnol. J. 12. 35.

Valdez, B. C., French, B. A., Younathan, E. S., Chang, S. H. (1989) Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase. J. Biol. Chem. 264, 131-135.

36.

Xu, P., Li, L., Zhang, F., Stephanopoulos, G., Koffas, M. (2014) Improving fatty acids production by engineering dynamic pathway regulation and metabolic control. Proc. Natl. Acad. Sci. USA. 111, 11299-11304.

37.

Kim, S., Lee, C. H., Nam, S. W., Kim, P. (2011) Alteration of reducing powers in an isogenic phosphoglucose isomerase (pgi)-disrupted Escherichia coli expressing NAD(P)-dependent malic enzymes and NADP-dependent glyceraldehyde 3-phosphate dehydrogenase. Lett. Appl. Microbiol. 52, 433-440.

38.

Qi, H., Li, S., Zhao, S., Huang, D., Xia, M., Wen, J. (2014) Model-driven redox pathway manipulation for improved isobutanol production in Bacillus subtilis complemented with experimental validation and metabolic profiling analysis. PLoS One. 9, e93815.

39.

Xue, G.-P., Johnson, J. S., Dalrymple, B. P. (1999) High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J. Microbiol. Meth. 34, 183–191.

40.

Yan, X., Yu, H. J., Hong, Q., Li, S. P. (2008) Cre/lox system and PCR-based genome engineering in Bacillus subtilis. Appl. Environ. Microbiol. 74, 5556-5562.

41.

Salis, H. M. (2011) The ribosome binding site calculator. Methods Enzymol. 498, 19-42.

42.

Yang, S., Du, G., Chen, J., Kang, Z. (2017) Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis. Appl. Microbiol. Biotechnol. 101, 4151-4161.

43.

Mengin-Lecreulx, D., van Heijenoort, J. (1996) Characterization of the essential gene glmM

44.

Badet, B., Vermoote, P., Haumont, P. Y., Lederer, F., Goffict, F. L. (1987) Glucosamine

encoding phosphoglucosamine mutase in Escherichia coli. J. Biol. Chem. 271, 32–39. synthetase from Escherichia coli: purification, properties, and glutamine-utilizing site location. Biochemistry. 26, 1940-1948.

27 28

30


Page 30 of 39

Page 31 of 39 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


1 2

(Figure 1)

3 31



1 2

(Figure 2)

3

32


Page 32 of 39

Page 33 of 39 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


1 2

(Figure 3)

33



1 2

(Figure 4)

34


Page 34 of 39

Page 35 of 39 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


1 2

(Figure 5)

35



1 2

(Figure 6) 36


Page 36 of 39

Page 37 of 39 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


1 2

(Figure 7)

37



1 2

(Figure 8)

3

38


Page 38 of 39

Page 39 of 39 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


1 2

Graphical Abstract

39


Engineering a Glucosamine-6-phosphate Responsive glmS Ribozyme

Recommend Documents