Corynebacterium glutamicum Metabolic Engineering with CRISPR

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Corynebacterium glutamicum metabolic engineering with CRISPR interference (CRISPRi) Sara Cleto, Jaide K Jensen, Volker F. Wendisch , and Timothy K. Lu ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.5b00216 • Publication Date (Web): 01 Feb 2016 Downloaded from http://pubs.acs.org on February 2, 2016

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ACS Synthetic Biology

Corynebacterium glutamicum metabolic engineering with CRISPR interference (CRISPRi) 1,2§

Sara Cleto, 1,2,3§Jaide VK Jensen, 3Volker F Wendisch, & 1,2Timothy K Lu

1

Department of Electrical Engineering & Computer Science and Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States of America 2

MIT Synthetic Biology Center, 500 Technology Square, Cambridge, Massachusetts 02139, United States of America

3

Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University, Bielefeld, Germany

§

These authors contributed equally

18 19

Abstract

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Corynebacterium glutamicum is an important organism for the industrial

21

production of amino acids. Metabolic pathways in this organism are usually

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engineered by conventional methods such as homologous recombination, which

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depends on rare double-crossover events. To facilitate the mapping of gene

24

expression levels to metabolic outputs, we applied CRISPR interference

25

(CRISPRi) technology using deactivated Cas9 (dCas9) to repress genes in C.

26

glutamicum. We then determined the effects of target repression on amino acid

27

titers. Single-guide RNAs directing dCas9 to specific targets reduced expression

28

of pgi and pck up to 98%, and of pyk up to 97%, resulting in titer enhancement

29

ratios of L-lysine and L-glutamate production comparable to levels achieved by

30

gene deletion. This approach for C. glutamicum metabolic engineering, which

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only requires three days, indicates that CRISPRi can be used for quick and

32

efficient metabolic pathway remodeling without the need for gene deletions or

33

mutations and subsequent selection.

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ACS Paragon Plus Environment

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Keywords: C. glutamicum, CRISPRi, sgRNA/dCas9, amino acid, metabolic

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engineering

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Introduction

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For the past 50 years, the industrialized world has relied on the extraordinary

40

ability of the soil organism Corynebacterium glutamicum to synthesize and

41

secrete copious amounts of amino acids 1–3. Molecules generated via C.

42

glutamicum fermentation are used as components in animal feed, nutritional

43

supplements, cosmetics, flavor enhancers, and the synthesis of pharmaceuticals

44

4–6

45

are projected to reach a market size of US $20.4 billion by 2020 7.

. These products rank amongst the most important industrial bioproducts and

46 47

Early efforts to increase amino acid production by C. glutamicum involved

48

random chemical or ultraviolet DNA mutagenesis 1,8. Despite their stronger

49

producer phenotypes, the mutagenized bacteria, initially genetically

50

uncharacterized, were often genetically unstable or had growth defects 1,9,10. With

51

the advancement of molecular genetics and whole genome sequencing, methods

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of rational metabolic engineering could be applied: specific genes could be

53

knocked out or introduced into the genetic makeup of an organism to enhance its

54

production of the desired metabolites 11. Genetic modifications can be introduced

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in C. glutamicum by transposon mutagenesis, homologous recombination, and

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recombinase-mediated deletions. Transposable elements can be randomly

57

inserted into the C. glutamicum genome 9, but precise gene modifications rely on

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the integration of suicide vectors, which can be followed by a second

59

recombination event to remove the plasmid backbone.

60 61

However, these genetic engineering tools are not very efficient. Suicide vectors

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are integrated into the bacterial genome with an efficiency of 102 mutants per µg

63

of DNA, of which 98% correspond to Campbell-like integrations and 2%

64

correspond to double-crossover events 12. SacB counter-selection facilitates


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screening for double-crossover events: for example, out of 2 x 106 cells having a

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single-crossover event, 200 (0.01%) grew on sucrose, 56 of which had

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undergone a second recombination that excised the plasmid backbone only 13.

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Others have reported integration efficiencies (Campbell-like) of 2.4 x 102 per µg

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of non-methylated plasmid DNA 14. These integrative vectors can be engineered

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to overexpress genes, by using constitutive or inducible promoters such as P180

71

(constitutive), Ptac (inducible if LacIq present), Ptrc (inducible if LacIq present), and

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PprpD2 (inducible) 9,15,16. The recombinase-based Cre/loxP system has further

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enabled the deletion of chromosomal regions 9,17,18. This efficient two-component

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system requires the integration of two loxP sites in the same orientation by

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homologous recombination. Once the sites are integrated so as to delimit the

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locus of interest and Cre recombinase is expressed, the DNA between the two

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loxP sites is efficiently excised 19.

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Systems biology has made considerable contributions to our understanding of

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the underlying intricacies and interrelationships of metabolic pathways 2,20,21 and

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may enable the predictive design of strains that meet well-defined specifications

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22–26

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one or more gene functions could be quickly and easily confirmed, the design-

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build-test cycle for the metabolic engineering of C. glutamicum could be

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accelerated. For example, it can be useful to construct derivative strains in which

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the expression of particular genes is reduced rather than knocked-out. The

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deletion of gltA in lysine-producing C. glutamicum rendered it unable to grow on

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glucose; however, this deletion resulted in suppressor mutants producing

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increased levels of L-lysine 27. This led to another study where laborious

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promoter tuning to identify optimal levels of gltA expression for L-lysine

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production by C. glutamicum was performed, resulting in the highest reported L-

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lysine yield at that time 28. Using existing approaches, it has been technically

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more difficult to generate mutants with reduced gene expression than those

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entirely lacking the expression of a particular gene (i.e., knockout mutants) 29.

. If the predicted phenotype resulting from the loss or reduced expression of

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In other organisms, tools are available for perturbing gene expression in a

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targeted fashion 30. For example, small regulatory RNAs (sRNA) have been used

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to identify and modulate the expression of genes of interest in Escherichia coli.

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Via combinatorial knockdown of several genes, the capacity of E. coli to produce

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tyrosine was approximately doubled 31, and E. coli strains having a 55% increase

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in cadaverine production were identified by using a library of synthetic sRNAs 31.

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This system, which works at the translational level, consists of a scaffold

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sequence and a target-binding sequence. The scaffold sequence is responsible

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for recruiting the RNA chaperone Hfq, which is required for the annealing of

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sRNA to its mRNA target, facilitating the degradation of that specific mRNA32.

106 107

Although Hfq homologues have been found in more than 50% of sequenced

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bacteria, C. glutamicum does not contain one 33,34. In fact, knowledge of sRNAs

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in Actinobacteria is very limited. Only recently have putative sRNA genes been

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identified in C. glutamicum (over 800 were found by using a high-throughput

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sequencing approach 34). Despite some advances 35, it is not fully understood

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how this sRNA regulatory mechanism functions in C. glutamicum. Further studies

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are thus required to determine the suitability of using synthetic sRNAs for

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modulating gene expression in this organism, as has been done with E. coli.

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To understand how modifying the expression of specific genes in C. glutamicum

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affects the titer of a particular bioproduct or alters pathway flux 36,37, a quick,

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reliable and easy approach is needed. The deactivated version of the Clustered

119

Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9) 38,39

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system, also known as CRISPR/dCas9 or CRISPR interference, offers a way to

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manipulate expression levels of specific genes 40,41. Using the Streptococcus

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pyogenes Cas9 protein, a single guide RNA (sgRNA) sequence is sufficient to

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direct Cas9 binding to specific DNA targets with a protospacer adjacent motif

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containing a GG dinucleotide 42. As opposed to the active Cas9 nuclease, the

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deactivated Cas9 protein (dCas9) used in CRISPRi lacks nuclease activity.

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dCas9 nonetheless retains the capacity to complex with the sgRNA and bind to


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the homologous locus. Instead of cleaving the DNA, this complex sterically

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blocks the progression of RNA polymerase and inhibits transcription. This

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process does not introduce permanent DNA-encoded mutations and thus

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phenotypes induced by CRISPRi can be reversed by shutting the CRISPRi

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system off 40,43.

132 133

Here, we report the application of CRISPRi to C. glutamicum, and its usefulness

134

in modulating the production of the amino acids L-lysine and L-glutamate via

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metabolic pathway regulation. CRISPRi provides a way to bypass the traditional

136

process of creating genetic mutants, which can be time-consuming and

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cumbersome. Ultimately, CRISPRi should enable the scalable and combinatorial

138

targeting of multiple genes, the mapping of gene expression pathways to

139

biosynthesis, and the enhanced productivity of C. glutamicum.

140 141 142

Results and Discussion

143

CRISPRi-based rfp repression

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To analyze the utility of CRISPRi for gene repression in C. glutamicum, we first

145

integrated the gene coding for a red fluorescent protein (rfp) into its chromosome.

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We chose to target both the template (T) and non-template (NT) strands of rfp as

147

there are contradictory reports regarding the efficiency of T-targeting at

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repressing transcription. Some authors have reported that T-targeting is

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inefficient 40,42, whereas other have found T-targeting to lead to effective

150

transcriptional repression 41,44. Inducing the expression of a plasmid-borne dcas9

151

with sodium propionate (using the PprpD2 promoter) in cells expressing sgRNAs

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targeted at the template (T) or non-template (NT) strand of the rfp gene resulted

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in reduced RFP production (supplemental figure 1). Surprisingly, adding sodium

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propionate to the strain expressing dcas9 alone (i.e., no sgRNA) resulted in

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higher levels of rfp expression.

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CRISPRi-based regulation of amino acid production by C. glutamicum

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Having seen that a heterologous gene (rfp) could be repressed by CRISPRi in C.

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glutamicum, we used this tool to target three endogenous genes of commercial

160

relevance: pgi, pck and pyk. The deletion of pgi leads to NADPH overproduction

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through the pentose-phosphate pathway and this results in increased L-lysine

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titers 23,45. The deletion of pck or pyk indirectly increases L-glutamate production

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24,46

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production through an enhanced anaplerotic flux 24,47. Disrupting pck also results

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in an accumulation of L-glutamate, via an enhanced flux towards oxaloacetate in

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the TCA cycle, due to the disruption of backward flux from oxaloacetate to

167

phosphoenolpyruvate 46.

. The disruption of pyk is thought to result in increased L-glutamate

168 169

To repress these genes, we built several sgRNAs that together with dCas9 would

170

sterically block transcription. In initial experiments, we found that producing

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dCas9 from an unrepressed Ptac promoter resulted in no clones after

172

transformation (data not shown). Previous work showed that other bacterial hosts

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in which dCas9 was expressed grew poorly or not at all 44,48. To overcome this

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issue, we repressed sgRNA and dcas9 expression from Ptac via the LacI

175

transcription factor, which can be induced by the addition of IPTG. These

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constructs were located on independent replicative plasmids: sgRNAs from

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pAL374 (Table 1), and dCas9 from plasmid pZ8-1 (Table 1). The following

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experiments were performed in CgXII minimal medium with 2% (w/v) glucose as

179

carbon source. In this section, sampling for amino acid quantification was

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performed immediately after glucose depletion with cells in stationary phase (the

181

standard for quantification of maximal amino acid production 49,50).

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When the CRISPRi system targeted the NT strand of pgi, the titer of L-lysine (p=

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4.65 x 10-13) increased by a factor of 2.1 over its sgRNA-less counterpart (Table

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2; Figure 1), suggesting strong repression of Pgi production. A milder effect (a

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1.05-fold increase over the control strain with dCas9 but without the sgRNA) was

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observed when the sgRNA targeted the T strand of the same gene (p=0.048).


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The increase in L-lysine production was much stronger when targeting the NT

189

versus the T strand (p= 2.31 x 10-12). In the absence of dCas9, expression of the

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sgRNAs alone did not interfere with the production of L-lysine (Figure 1c).

191 192

For pck, targeting either the T or NT strands resulted in increased glutamate

193

production by ~2.2-fold compared with a similar strain that only lacked the

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sgRNA (Figure 2; pT=0.02; pNT=0.006). No significant difference was observed

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between targeting the T or NT strand (Figure 2; p=0.90). For pyk, targeting the T

196

strand increased L-glutamate production by ~1.9 fold (p=0.03) and targeting the

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NT strand increased it 3.2-fold compared with the control strain carrying the

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dCas9 but no sgRNA (Figure 3; p=0.002). Similar to pgi, there was a statistically

199

significant difference between the fold-induction achieved by targeting the T

200

versus the NT strand of pyk, but the magnitude of the difference was smaller

201

(Figure 3; p=0.05). In the absence of dCas9, expression of the sgRNAs alone did

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not interfere with the production of L-glutamate (Figure 2c and 3c).

203 204

The increased L-lysine production we observed by repressing pgi with CRISPRi

205

is similar to that observed when pgi is deleted (Table 2) 45. The repression of pck

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and pyk expression via CRISPRi achieved L-glutamate production ratios

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(targeted/non-targeted) that exceed published results for the deletion of those

208

genes (Table 2) 24,45,46. However, the absolute amounts of amino acids produced

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using the CRISPRi system were not as high as when the genes were deleted

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24,45,46

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because of residual enzyme expression due to incomplete repression by

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CRISPRi.

, which could be due to the use of different strains or conditions, or

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Quantification of the mRNA levels of genes targeted by CRISPRi

215 216

We next evaluated the levels of transcription of each gene targeted by CRISPRi

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(Figure 4). Here, we sampled C. glutamicum cultures in mid-exponential phase


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(maximal growth rate) to estimate mRNA levels and concomitant amino acid

219

production. We normalized mRNA levels of the targeted genes to those of 16S

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rRNA and calculated grams of amino acid produced per gram of cell dry weight,

221

to account for the slight variations between the ODs of the strains and

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independent experiments, at the time of sampling.

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No CRISPRi-mediated repression of transcription was observed during the

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exponential phase when the T strand of pgi was targeted; however, targeting the

226

NT strand strongly repressed mRNA levels (Figure 4a, Table 2). The relative

227

levels of mRNA were reduced by nearly 98% (p=1.02x10-13), resulting in a 1.31-

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fold increase in L-lysine/gCDW, versus the control strain, which expressed

229

dCas9 but lacked the sgRNA (p=3.40x10-8). When the T strand of pck was

230

targeted, a 69% (p=2.04x10-7) reduction in its mRNA levels and a 1.28-fold

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increase in the levels of secreted L-glutamate/gCDW (p=0.02) were observed

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over the control levels of the strain lacking the sgRNA only (Figure 4, Table 2). In

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comparison, transcriptional repression was much higher when dCas9 was

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directed against the NT strand of pck (98% reduction in mRNA versus the

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sgRNA-less counterpart, p=1.13x10-14); this repression appeared to result in

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1.46-fold more L-glutamate/gCDW compared to the control strain. Despite the

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strong transcriptional repression (Figure 4b), the variability of the experimental

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data indicated that the increase in L-glutamate/gCDW in the exponential phase

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was non-significant at the p=0.08 level, which contrasts with the data obtained in

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stationary phase shown in Figure 2c. Finally, targeting the T and NT strands of

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pyk with CRISPRi resulted in mRNA reductions of 84% (pT=4.35x10-5) and 97%

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(pNT=0.0002) and 1.97- (pT=0.016) and 3.04- (pNT=0.0005) fold increases in

243

secreted L-glutamate/gCDW, respectively (Figure 4, Table 2), versus the control

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strain. In general, greater mRNA repression correlated with higher levels of

245

secreted amino acids per gram of cell dry weight.

246 247

Quantification of Enzymatic Activity

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Given that sgRNA/dCas9 did not fully repress transcription, we decided to

250

investigate enzyme activity levels in mid-exponential cultures of C. glutamicum in

251

which CRISPRi was present or not. These samples were identical to those taken

252

for mRNA quantification in Figure 4. The specific enzymatic activities of Pgi, Pck,

253

and Pyk reflect the amount of enzyme available in the cell at the time of

254

sampling. Pgi activity levels decreased by 95% in comparison with a control with

255

no sgRNA when the NT strand was targeted (p=1.25x10-38, Figure 5a, Table 2).

256

Targeting the T strand of pgi had no impact on the enzyme activity levels

257

observed (p=0.80, Figure 5a, Table 2), versus a control with no sgRNA.

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Targeting the NT strand in the absence of dCas9 had no effect on Pgi activity

259

(0.49 U/mg; p=0.20 versus strain with the same sgRNA and no dCas9). Pck

260

activity levels decreased by 49% (p=0.005) and 83% (p=9.08x10-7) when the T

261

and NT strands were targeted by specific sgRNAs, respectively, compared with a

262

control with no sgRNA (Figure 5b, Table 2). For Pyk, targeting the T or NT

263

strands resulted in a 26% (p=5.38x10-11) and 52% (p=1.85x10-24) decrease in

264

specific activity, respectively, compared with the no-sgRNA control (Figure 5c,

265

Table 2). As expected, there was a correlation between reductions in mRNA

266

levels (Figure 4) and the corresponding enzyme activity (Figure 5), with sgRNAs

267

directed against the NT strand leading to lower mRNA and enzyme activity levels

268

compared with those directed against the T strand.

269 270

Conclusion

271

Given its simplicity of design and ease of deployment, the CRISPR/Cas system

272

has enabled researchers to edit DNA or gene transcription levels (sgRNA/dCas9

273

or CRISPRi) across an extremely diversified range of organisms 51–53. The goal

274

of our study was to test the performance and suitability of CRISPRi for pathway

275

engineering in C. glutamicum. Our approach, involving the use of sgRNAs 42 and

276

the dCas9 enzyme to repress the expression of targeted genes 54, allowed us to

277

go from the initial cloning in E. coli to the final engineered C. glutamicum strains

278

ready for testing in as little as 3 days. The use of dCas9 instead of Cas9 omits


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the need to select for rare Cas9-mutated strains 55 or single- or double-crossover

280

mutants obtained via suicide vectors.

281 282

C. glutamicum plays a multi-billion dollar role in the production of two amino

283

acids with the largest market sizes, L-lysine for animal feed and L-glutamate for

284

food additives. The genes chosen for the present study, pgi, pck and pyk,

285

indirectly impact the production of these two amino acids. For pck and pyk, we

286

found that the repression level caused by the steric blockage of the progression

287

of the RNA polymerase to be equally efficient whether dCas9 annealed onto the

288

T or NT strand of the coding region. For all three genes, the amino acid

289

production measured after glucose depletion in early stationary phase increased

290

in a statistically significant manner when gene repression was mediated by any

291

of the sgRNAs in the presence of dCas9 (Figure 1-3). In most cases, targeting

292

the NT strand with the CRISPRi system resulted in greater transcriptional

293

repression and amino acid production than targeting the T strand. This

294

observation is consistent with earlier publications that reported that template-

295

strand targeting can be inefficient 40,42, even though other groups have shown

296

that template-strand repression can be achieved 41,44.

297 298

Due to its programmability and ease of implementation, we envision that

299

CRISPRi-based gene repression could be used to identify putative genes that

300

should be targeted for enhancing bioproduction. Unlike knockout-based

301

approaches to metabolic engineering, the likelihood of completely inhibiting gene

302

expression with CRISPRi is low. One could take advantage of this property to

303

map how intermediate levels of gene expression translate to metabolic

304

production. Furthermore, multiplexed gene perturbation via the expression of

305

multiple sgRNAs from barcoded constructs (e.g., those built using CombiGEM

306

assembly 56) coupled with high-throughput characterization via liquid

307

chromatography coupled to mass spectrometry could be used for combinatorial

308

metabolic pathway engineering. However, in other cases, even a low expression

309

level of certain metabolic genes could potentially contribute enough enzyme


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310

activity to maintain flux at a near wild-type level and thus mask the effect of

311

targeting such genes. In the future, dynamic metabolic pathways could be

312

engineered by coupling the expression of CRISPRi system to promoters that are

313

responsive to metabolite concentration. The ability to tune metabolic pathways in

314

response to intracellular or extracellular conditions may help to maximize

315

production titers for the molecules of interest 57,58.

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Table 1. Strains and plasmids used in this study

Strain

Properties

Source

C. glutamicum ATCC 13032

Wild type, biotin auxotroph

C. glutamicum DM1729 (DSM17576)

DM1729 is an aminoethylcysteine-resistant mutant of ATCC 13032; pyc(P458S) hom(V59A) lysC(T311I) - L - Lysine overproducer

Evonik Degussa GmbH

E. coli DH5α

Cloning strain

Lab stock

Plasmids

ATCC

Properties

Source

59

pDSW204

E. coli IPTG inducible expression vector, AmpR

pZ8-1

E. coli – C. glutamicum Ptac constitutive expression shuttle vector, KanR

pAL374

E. coli – Corynebacterineae expression shuttle vector, SpecR

pZ8-T_dCas9

pZ8-1 plasmid carrying dcas9, driven by the IPTG-inducible Ptac promoter, KanR

This study

pZ8-P_dCas9

pZ8-1 plasmid carrying dcas9 driven by the propionate-inducible prpD2 promoter (PprpD2), KanR

This study

pZ8-Ptac

IPTG inducible version of pZ8-1 plasmid, to which lacIq was added, KanR

This study

pZ8-Prp

pZ8-1 with the PprpD2 propionate-inducible promoter instead of Ptac, KanR

This study

pPP208

Donor of dcas9

pAL-pgi_T

pAL374 plasmid carrying the pgi (T) sgRNA, targeting the template strand of pgi, SpecR

This study

pAL-pgi_NT

pAL374 plasmid carrying the pgi (NT) sgRNA, targeting the non-template strand of pgi, SpecR

This study

pAL-pck_T

pAL374 plasmid carrying the pck (T) sgRNA targeting, the template strand of pck, SpecR

This study

pAL-pck_NT

pAL374 plasmid carrying the pck (NT) sgRNA, targeting the non-template strand of pck, SpecR

This study

pAL-pyk_T

pAL374 plasmid carrying the pyk (T) sgRNA targeting the template strand of pyk, SpecR

This study

pAL-pyk_NT

pAL374 plasmid carrying the pyk (NT) sgRNA targeting the non-template strand of pyk, SpecR

This study

pRIM2

E.coli – C. glutamicum shuttle vector, integrative in C. glutamicum downstream of ppc, KanR

pRIM3

GentR version of pRIM2

Evonik Degussa GmbH 60

54


61

This study

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pRIM3-rfp

pRIM3 carrying rfp GentR

This study

pBTK30

Donor of GentR

pZ8-1_rfp

Donor of rfp, KanR

pAL-rfp_NT

pAL374 plasmid carrying the rfp (NT) sgRNA targeting the non-template strand of rfp, SpecR

This study

pAL-rfp_T

pAL374 plasmid carrying the rfp (T) sgRNA targeting the template strand of rfp, SpecR

This study

62

Sinskey Lab (MIT)

319 320

Table 2. Ratio of changes in amino acid production, mRNA levels and

321

enzymatic activity in mid-exponential phase cultures of C. glutamicum

322

when targeting genes with CRISPRi §.

Gene targeted (ID)

Strand targeted

Amino acid ratio (after glucose depletion in stationary phase) CRISPRi

T

1.05

Amino acid ratio (exponential phase)

Enzyme activity ratio (exponential phase)

1.12

1.01

1.01

0.02

1.31

0.05

0.31

1.28

0.51

0.02

1.46

0.17

0.16

1.97

0.74

0.03

3.04

0.48

1.7 45

pgi

pck

Gene deletion (reference values)

mRNA ratio (exponential phase)

NT

2.14

T

2.18 4.5 46 §§

NT

2.24

T

1.92 1.25 24 §§§

pyk NT

3.25

323 324 325 326 327 328 329 330 331

§

Results are expressed as the ratio between (dCas9+sgRNA strain) / (dCas9+no sgRNA strain).

§§

In the study referenced, Tween 60 was used to induce glutamate production, whereas

ethambutol was used in our paper. §§§

In the study referenced, biotin was used to induce glutamate production, whereas ethambutol

was used in our paper. T = template; N = non-template. ND – Not determined.

332 333


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Page 14 of 35

334

Table 3 Primers used for the quantification of the mRNA in the samples by

335

qRT-PCR.

336 Template

Strand

Primer sequence (5’ – 3’)

F

TCATTGGTTTCGCTCGTCCA

R

AGCGTTCTTACCGAAAGCCA

F

ATTGGCTACAACGCTGGTGA

R

CCACTTCAGAACGCGAGAGT

F

GATACCGCAAAGCGTGTGG

R

GACAGGTGGACACAGGAAGG

F

TTACCTGGGCTTGACATGGAC

R

GCTGGCAACATAAGACAAGGG

pgi

pck

pyk

16S rRNA

337 338 339

Materials and Methods

340

Microorganisms, plasmids and growth conditions

341 342

Bacterial strains and plasmids are listed in Table 1.

343 344

The plasmid pRIM3_rfp was assembled from pRIM2 by the Isothermal Assembly

345

Method 63. The original kanamycin resistance cassette (same as in pZ8-Prp) was

346

replaced with a gentamycin cassette and the rfp gene was cloned from pZ8-

347

1_rfp, together with the Ptac promoter and T1 terminator.

348 349

The plasmid pZ8-Ptac, which is an inducible version of plasmid pZ8-1, was

350

obtained by cloning lacIq from pDSW405 upstream of the Ptac promoter by the

351

Isothermal Assembly Method.

352 353

The plasmid pZ8-Prp is pZ8-1 with Ptac replaced by the propionate-inducible

354

promoter PprpD2 (Table 1). The promoter was ordered as a gBlock from Integrated


14

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355

DNA Technologies (IDT, USA) and cloned into pZ8-1 lacking Ptac by the

356

Isothermal Assembly Method.

357 358

The gene coding for a nuclease-inactivated version of Cas9 - dcas9 - from

359

Streptococcus pyogenes was cloned from plasmid pPP208 into the C.

360

glutamicum replicative plasmids pZ8-Ptac and pZ8-Prp, under the control of an

361

IPTG- or a propionate-inducible promoter, respectively.

362 363

The short, synthetic version of the sgRNA was designed to contain a 24-bp

364

region of homology to the transcriptional template (T) or non-template (NT)

365

strands of the target DNA including the seed sequence, followed by the dCas9

366

handle and the S. pyogenes terminator. The expression of this sgRNA was

367

driven by a Ptac, ordered as a gBlock from IDT and cloned into the replicative

368

plasmid pAL374. The base-pairing regions, handle and terminators used were

369

the same as those previously described for the sgRNA 40. The target sequences

370

are listed in Figures 1, 2 and 3. All sgRNAs were cloned into pAL374 by the

371

Isothermal Assembly Method, the plasmid backbone having been amplified using

372

the primers listed in Table 2, which also removed Ptrc.

373 374

All plasmids were introduced by transformation into E. coli DH5α and maintained

375

in that strain, grown in Lysogeny (Miller, LabExpress, USA) broth (for liquid

376

cultures) or agar (for plates, with 15 % agar, Apex) with the appropriate

377

antibiotics (Table 2), and incubated at 37°C.

378 379

C. glutamicum strains were also maintained on LB agar plates, supplemented

380

with the appropriate antibiotic when necessary (Table 1), but incubated at 30°C.

381

Liquid cultures were prepared in Brain-Heart Infusion (BHI, Sigma-Aldrich,

382

Germany) medium. Electrocompetent cells were prepared as described

383

elsewhere 64.

384 385

All strains were kept as glycerol stocks at -80°C, in 30% glycerol LB broth.


15


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Page 16 of 35

386 387

Integration of pRIM3_RFP in C. glutamicum

388 389

pRIM3_RFP is a suicide vector that integrates into the host chromosome by a

390

single crossover event downstream of ppc, which has been previously described

391

to be a safe harbor 61. After electroporation and integration of this plasmid, the

392

insertion can be phenotypically screened, as in addition to the acquired

393

gentamicin resistance, the colonies having the insertion exhibit a red color.

394 395

CRISPRi-based regulation of RFP expression in C. glutamicum

396 397

sgRNAs were designed to target the T or NT strands of rfp within its first 150 bp

398

(Supplemental Figure 1a). For the rfp repression assay, the expression of dcas9

399

was driven by a propionate-inducible promoter. Overnight cultures without

400

inducer were used as inocula for new cultures with and without inducer. This

401

experiment was performed three times with two biological replicates.

402 403

All strains were grown in LB supplemented with 2 g/L of sodium propionate, at

404

30°C for 48 hr. Aliquots of all cultures were diluted 1:100 in Phosphate Buffered

405

Saline (pH=7) and run in a BD-FACS LSR Fortessa cell analyzer (BD

406

Biosciences, CA). The fluorescence was measured using a Texas-Red filter with

407

a 561 nm excitation laser and a 610/20 nm emission filter. For each sample,

408

50,000 cells were analyzed and gated using forward scatter and side scatter.

409 410

CRISPRi-based regulation of amino acid production by C. glutamicum

411 412

Genes pgi (AGT04848.1), pck (AGT06569.1) and pyk (AGT05824.1), coding for

413

enzymes whose absence leads to an increased production of L-lysine (pgi) and

414

L-glutamate (pck and pyk), were selected as individual targets for repression by

415

the sgRNA/dCas9 complex. The sgRNAs were designed to anneal onto the first

416

150 bp of the T and NT strands of genes pgi, pck or pyk (Supplemental Figure 2).


16

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417 418

Prior to every amino acid production experiment (Figure 1-3), C. glutamicum cells

419

from glycerol stocks were streaked onto LB agar plates with the appropriate

420

antibiotics and incubated at 30°C overnight. These cells were subsequently used

421

to inoculate BHI broth. Upon overnight growth, the uninduced precultures were

422

harvested by centrifugation (4000 rpm, 5 min, 4°C) and washed with 0.9% NaCl.

423

Ten mL of 2% (w/v) glucose CgXII minimal medium 65, supplemented with 30

424

µg/L protocatechuic acid and 1 mM of the dcas9 inducer IPTG were inoculated to

425

an optical density (OD) of 1 at 610 nm. When appropriate, kanamycin and/or

426

spectinomycin were added to a final concentration of 25 µg/mL and 100 µg/mL,

427

respectively.

428 429

Amino acids are typically quantified in the early stationary phase, when glucose

430

is depleted and the concentration of amino acids peaks due to their accumulation

431

over time 50,66. The cultures were grown in 125 mL flasks at 30°C, with a shaking

432

frequency of 200 rpm, until glucose was depleted, as measured by Quantofix®

433

Glucose strips (Machery-Nagel, Germany).

434 435

C. glutamicum ATCC 13032 (Table 1) was used for L-glutamate production. It

436

required supplementing CgXII with 20 mg/L ethambutol, which stimulates L-

437

glutamate secretion 67.

438 439

C. glutamicum DM1729 (Table 1) is an S-aminoethyl-L-cysteine-resistant mutant

440

of C. glutamicum ATCC 13032, previously developed for the specific purpose of

441

L-lysine overproduction. It did not require any additional supplements.

442 443

Growth of all cultures was monitored over time by measuring their OD with an

444

Infinite 200 Pro microplate reader (Tecan, Switzerland). Three independent

445

experiments were performed in triplicate. The OD610nm conversion to cell dry

446

weight (CDW) followed the ratio previously described of OD610nm1 = 0.25 g CDW

447

68

.


17


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Page 18 of 35

448 449

Quantification of the mRNA levels of genes targeted by CRISPRi

450 451

In order to determine the level of CRISPRi-based transcriptional repression,

452

quantitative reverse transcription PCR (qRT-PCR) was performed on total RNA

453

samples (Figure 4). This analysis was performed with cells in mid-exponential

454

phase. One-mL aliquots of each culture were taken at mid-exponential phase for

455

total RNA extraction and amino acid quantification.

456 457

Aliquots were centrifuged at 17,000 rpm for 15 seconds, and the pellets were

458

immediately flash frozen and stored at -80°C until further processing. The cells

459

were lysed by resuspension of pellets in RA1 buffer and bead beating for 2 x 15

460

seconds (Mini-Beadbeater-16, Biospec Products, USA) with intermittent cooling

461

on ice. The steel beads used were RNase-free and 0.2 mm in diameter (KSE

462

Scientific, USA). Total RNA was extracted using the Illustra RNAspin Mini Kit

463

according to the recommendations of the manufacturer (GE Life Sciences, UK).

464

To remove residual co-extracted DNA, the purified RNA was treated with DNase

465

(Turbo DNA-free kit, Life Technologies, USA).

466 467

qRT-PCR was performed using the KapaTM SYBR®Fast One-Step qRT-PCR kit

468

(Kapa Biosystems, USA), the LightCycler® 96 System (Roche, USA), 50 ng of

469

total RNA extracted and the primers indicated in Table 3. Results were analyzed

470

following the Livak method 69, using the threshold cycle of 16S rRNA for

471

normalization 70.

472 473

Enzyme activity assays

474 475

To directly investigate the actual amounts of enzyme being made (Figure 5), 9

476

mL aliquots of mid-exponential C. glutamicum cultures were collected

477

concomitantly with those for mRNA measurements (i.e., at mid-exponential

478

phase). They were then centrifuged at 4000 rpm for 5 min at 4°C, and the pellets


18

Page 19 of 35

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479

were immediately flash frozen and stored at -80°C until they underwent further

480

processing.

481 482

Cell pellets used for the Pgi enzyme assay were resuspended in 50 mM

483

triethanolamine (TEA) at pH 7.2. Cell pellets used for the Pck and Pyk enzyme

484

assays were resuspended in a solution consisting of 20 mM KCl, 5 mM MnSO4, 2

485

mM DTT, 0.1 mM EDTA, 30% glycerol and 100 mM Tris-HCl at pH 7.5. The cells

486

were then lysed by a 6-minute, 0.5 cycle and 55 amplitude sonication (Sonopuls

487

GM 200, Bandelin electronic GmbH & Co KG, Germany) and centrifuged for 60

488

min at 4°C and 14,600 rpm. Supernatants were collected as crude extracts to

489

measure the glucose-6-phosphate isomerase, pyruvate kinase, and PEP

490

carboxykinase activities.

491 492

Glucose-6-phosphate isomerase activity was assayed as described by Lindner et

493

al 23. The formation of NADPH (ε(340 nm) = 6.3 mM-1 cm-1) was monitored at 340

494

nm.

495 496

Pyruvate kinase activity was assayed essentially as described by Netzer et al. 47.

497

The assay mixture contained 100 mM TEA buffer (pH 7), 15 mM MgCl2, 1 mM

498

ADP, 0.25 mM NADH, 12 mM PEP, 5 U L-lactate dehydrogenase, and between

499

5 and 10 µL of crude extract (corresponding to 0.075 and 0.15 mg of protein,

500

respectively). The decrease of NADH (ε(340 nm) = 6.3 mM-1 cm-1) was monitored

501

at 340 nm.

502 503

The activity of PEP carboxykinase was assayed as described by Klaffl et al. 71

504

with the following modifications: the assay mixture contained 100 mM HEPES

505

buffer, (pH 6.8), 0.4 mM NADH, 1 mM GDP, 50 mM NaHCO3, 2.5 mM PEP, 0.5

506

mM MnCl2, 10 mM MgCl2, 4 U malate dehydrogenase, and between 5 and 10 µl

507

of crude extract (corresponding to 0.075 and 0.15 mg of protein, respectively).

508

The decrease of NADH (ε(340 nm) = 6.3 mM-1 cm-1) was monitored at 340 nm.

509


19


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Page 20 of 35

510

One unit of enzymatic activity was defined as 1 mmol of NADPH formed per

511

minute (U/min) or 1 mmol of NADH consumed per minute (U/min), and the

512

calculation of the specific activity took into account the enzyme concentration in

513

each of the samples (U/mg).

514 515 516

Amino acid quantification

517 518

Samples for amino acid quantification were taken in mid-exponential phase

519

(mRNA vs. amino acid analysis, Figure 4) or directly after glucose depletion

520

(effect of CRISPRi on amino acid production, Figure 1-3).

521 522

The samples were centrifuged at 17,000 rpm for 15 seconds and automatically

523

derivatized using the Fluoraldehyde™ o-Phthaldialdehyde Reagent Solution

524

(Thermo Scientific, USA), prior to entering the chromatographic separation

525

column. Compounds were separated by high-pressure liquid chromatography

526

(HPLC, 1200 series, Agilent) with a RP18 column (Eclipse XDB-C18, 4.6 x 150

527

mm, Agilent) and a fluorescence detector (FLD G1321A, 1200 series, Agilent).

528 529

The mobile phases were 0.1 M sodium acetate at pH 7.2 (A) and 100% methanol

530

(B). The gradient used was as follows: 0 min 20% B, 0.5 min 38% B, 2.5 min

531

46% B, 3.7 min 65% B, 5.5 min 70% B, 6 min 75% B, 6.2 min 85% B, 9.7 min

532

20% B and 11.9 min 20%, at a flow rate of 1.2 mL/min.

533 534

Ornithine was used as the internal standard, and standard curves for determining

535

the amino acid concentrations in the supernatants were drawn by using solutions

536

of L-glutamate and L-lysine at known concentrations.

537 538

Statistical treatment of data

539


20

Page 21 of 35

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540

All results were tested for significance using the unpaired heteroscedastic

541

Student's T-Test. The level of significance of the differences observed between

542

the control (strain carrying plasmids pZ8-T_dCas9 and pAL374) and test

543

samples (strains carrying plasmids pZ8-T_dCas9 and pAL374 with an sgRNA)

544

was expressed as one, two or three stars, for *p≤ 0.05, **p≤ 0.01 and ***p≤

545

0.001, respectively. “NS” stands for non-significant, when p>0.05.

546 547 548 549

Acknowledgements

550

Jens Plassmeier and Anthony Sinskey for helpful discussions and C. glutamicum

551

plasmids, and Karen Pepper for editing. JVKJ wishes to acknowledge Augustinus

552

Fonden for financial support and Evonik Degussa GmbH for providing the L-

553

lysine overproducer strain. This work was supported by the National Institutes of

554

Health (DP2 OD008435, P50 GM098792), the Office of Naval Research

555

(N00014-13-1-0424), the National Science Foundation (MCB-1350625) and Wuxi

556

NewWay Biotech Ltd (Jiangsu Province International Collaboration Grant

557

#BZ2014014).

The authors thank Irene Krahn for the help with the Pck and Pyk enzyme assays,

558


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Page 22 of 35

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expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408.

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(71) Klaffl, S., Brocker, M., Kalinowski, J., Eikmanns, B. J., and Bott, M. (2013) Complex regulation of the phosphoenolpyruvate carboxykinase gene pck and characterization of its Gntr-type regulator IolR as a repressor of myo-inositol utilization genes in Corynebacterium Glutamicum. J. Bacteriol. 195, 4283–4296.

776 777 778 779

Supporting information

780 781

Supplemental Figure 1: The CRISPRi system efficiently represses chromosomally-inserted rfp.

782

Supplemental Figure 2: Loci for CRISPRi annealing on pgi, pck and pyk.

783 784 785

Figure Legends

786 787

Figure 1: CRISPRi efficiently represses pgi transcription, increasing L-

788

lysine production

789

(a) Central metabolic pathway. The notation in red represents the reduced

790

transcription of pgi due to CRISPRi-mediated repression. (b) Gene and

791

sequences targeted by dCas9. (c) Amino acid titers (g/L) of the control and test

792

strains (N=9 per strain) were determined upon glucose depletion. The error bars

793

represent the standard deviation of samples. *p≤ 0.05 and ***p≤ 0.001.

794 795

Figure 2: CRISPRi efficiently represses pck transcription, increasing L-

796

glutamate production

797


798

transcription of pck due to CRISPRi-mediated repression (b) Gene and


27


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Page 28 of 35

799


800


801

represent the standard deviation of samples. “NS” represents non-significant

802

(p>0.05), **p≤ 0.01 and ***p≤ 0.001.

803 804

Figure 3: CRISPRi efficiently represses pyk transcription, increasing L-

805

glutamate production

806


807

transcription of pgi due to CRISPRi-mediated repression. (b) Gene and

808


809


810

represent the standard deviation of samples. *p≤ 0.05, **p≤ 0.01 and ***p≤ 0.001.

811 812

Figure 4: mRNA levels decrease with CRISPRi targeting, resulting in

813

increases in amino acid levels.

814

In order to measure the mRNA levels of pgi, pck and pyk in the cells, samples

815

were collected in mid-exponential phase. Upon total RNA extraction, qRT-PCR

816

was performed on each of the 9 replicates (per strain) with technical replicates

817

and using 16S rRNA for data normalization. Amino acid titers were measured by

818

HPLC and are reported as grams of amino acid per cell dry weight. (a)

819

sgRNA/dCas9-mediated pgi targeting, (b) sgRNA/dCas9-mediated pck targeting

820

and (c) sgRNA/dCas9-mediated pyk targeting. “NS” represents non-significant

821

(p>0.05), *p≤ 0.05, **p≤ 0.01 and ***p≤ 0.001.

822 823

Figure 5: The specific Pgi, Pck and Pyk activities in crude extracts

824

decrease when the corresponding genes are targeted by the CRISPRi

825

system.

826

Crude extracts from mid-exponential growing strains (9 biological replicates per

827

strain, each with three technical replicates) were obtained upon cell lysis and

828

used for the quantification of the Pgi, Pck or Pyk activity, as seen by a decrease

829

in the NADH available. The specific activity (U/mg) of (a) Pgi, (b) Pck and (c) Pyk


28

Page 29 of 35

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


830

when CRISPRi targets the template (T) or non-template (NT) DNA strands is

831

shown. “NS” represents non-significant (p>0.05), ***p≤ 0.001.

832 833

Supplemental Figure 1: CRISPRi efficiently represses chromosomally

834

expressed RFP.

835

(a) Gene and sequences targeted by dCas9. (b) Schematic of the loci targeted

836

by the T and NT sgRNAs. The leading and lagging strands of the rfp are shown,

837

and the first base of its start codon is numbered as 1. The target sequences

838

(template and non-template strands) are highlighted in grey and the PAM

839

sequence in light grey. Their loci are denoted by the numbering. Mean RFP

840

levels (AU) measured by FACS of the control and test strains determined in

841

stationary phase (N=6 per strain). The error bars represent the standard

842

deviation of samples. “NS” represents non-significant (p>0.05), *p≤ 0.05 and

843

***p≤ 0.001.

844 845

Supplemental Figure 2: Loci for CRISPRi annealing on pgi, pck and pyk.

846 847

Schematic of the (a) pgi, (b) pck and (c) pyk loci targeted by the T and NT

848

sgRNAs. The leading and lagging strands of the gene are shown, and the first

849

base of its start codon is numbered as 1. The target sequences (template and

850

non-template strands) are highlighted in grey and the PAM sequence in light

851

grey. Their location is denoted by the numbering.

852


29

Glucose

Glucose 6-P

Ribulose 5-P

Pgi

Pgi Lysine ACS Synthetic Biology Page 30 of 35 Pck Glutamate Pyk Xylulose 5-P

Ribose 5-P

Fructose 6-P

Erythrose 4-P

Pfk Fructose 1, 6-DP

1 2 3 4 5 6

Dihydroxyacetone P

Fba Sedoheptulose 7-P Glyceraldehyde 3-P

Phosphoenolpyruvate

Pck

Pyk Pyruvate

Pyc

ACS Paragon RNApol Plus Environment sgRNA

Oxaloacetate

Ppc

dCas9

a

Glucose

Page 31 of 35

NADP+

Glucose 6-P

NADPH Ribulose 5-P

ACSPhosphate Synthetic Biology Pentose Pathway Xylulose 5-P

***

Lysine g/L

1 Pgi 2 Gluconeogenesis 3 Ribose 5-P Fructose 6-P 4 Erythrose 4-P 5 6 Pfk 7 8 Fructose 1, 6-DP 9 Fba 10 Sedoheptulose 7-P 11 12 DihydroxyGlyceraldehyde 3-P 13 acetone P 14 b 15 Phosphoenolpyruvate 16 Pck Pyk 17 18 Ppc 19 Pyruvate 20 c 21 3 Pyc L-Lysine 22 23 24 Oxaloacetate 25 2 26 27 28 1 29 TCA 30 Cycle 31 2-Oxoglutarate 0 32 Empty pAL374 sgRNA pgi (T) sgRNA pgi (NT) ACS Paragon Plus Environment 33 34 35

***

*

dCas9 + empty pAL374

dCas9 + sgRNA dCas9 + sgRNA pgi (T) pgi (NT)

a

Glucose

Glucose 6-P

ACSPhosphate Synthetic Biology Pentose Pathway Ribulose 5-P

Page 32 of 35

Xylulose 5-P

1 2 GlucoPgi 3 neogenesis Ribose 5-P 4 Fructose 6-P 5 Erythrose 4-P 6 Pfk 7 8 Fructose 1, 6-DP 9 10 Fba 11 Sedoheptulose 7-P 12 DihydroxyGlyceraldehyde 3-P 13 acetone P 14 b 15 Phosphoenolpyruvate 16 17 Pck Pyk 18 19 Ppc Pyruvate 20 c 21 22 Pyc 23 24 25 Oxaloacetate 26 27 28 29 TCA 30 Cycle 31 2-Oxoglutarate 32 33 ACS Paragon Plus Environment L-Glutamate 34 35 36

** *

NS

a

Glucose

Page 33 of 35

Glucose 6-P

ACSPhosphate Synthetic Biology Pentose Pathway Ribulose 5-P

Xylulose 5-P

1 2 GlucoPgi 3 neogenesis Ribose 5-P 4 Fructose 6-P 5 Erythrose 4-P 6 Pfk 7 8 Fructose 1, 6-DP 9 10 Fba 11 Sedoheptulose 7-P 12 DihydroxyGlyceraldehyde 3-P 13 acetone P 14 b 15 Phosphoenolpyruvate 16 17 Pck Pyk 18 19 Ppc Pyruvate 20 c 21 22 Pyc 23 24 25 Oxaloacetate 26 27 28 29 TCA 30 Cycle 31 2-Oxoglutarate 32 33 ACS Paragon Plus Environment L-Glutamate 34 35 36

**

*

*

Glutamate (g) / dry weight (g)

Glutamate (g) / dry weight (g)

NS

***

1.5

mRNA relative levels

1 0.4 2 3 0.2 4 5 6 0 7 8 9 b10 0.08 11 12 0.06 13 14 0.04 15 16 0.02 17 18 0 19 20 c21 22 0.15 23 24 250.1 26 27 0.05 28 29 30 0 31 32 33

*** 34 of 35 ACS Synthetic Biology Page

***

NS NS

***

0.5

1.00

dCas9 + dCas9 + dCas9 + empty sgRNA sgRNA pAL374 pgi (T) pgi (NT)

*** **

NS

dCas9 + dCas9 + dCas9 + empty sgRNA sgRNA pAL374 pyk (T) pyk (NT)

**

0.50

0.25

1.00

*

**

0.75

0.00

dCas9 + dCas9 + dCas9 + empty sgRNA sgRNA pAL374 pck (T) pck (NT)

***

NS

1.0

0.0

dCas9 + dCas9 + dCas9 + empty sgRNA sgRNA pAL374 pgi (T) pgi (NT)


Lysine (g) / dry weight (g)

0.6


a

dCas9 + dCas9 + dCas9 + empty sgRNA sgRNA pAL374 pck (T) pck (NT)

*** ***

0.75

0.50

***

0.25

0.00

dCas9 + dCas9 + dCas9 + ACS Paragon Plus Environment empty sgRNA sgRNA pAL374

pyk (T) pyk (NT)

ACS Synthetic Biology aPage 35 of 35 *** NS *** 1 2 3 4 5 6 7 8 b9 10 11 12 13 14 15 16 17 18 c19 20 21 22 23 24 25 26 27 28

***

*** ***

***

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Corynebacterium glutamicum Metabolic Engineering with CRISPR

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