Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Letter
CRISPR/Cas9-based efficient genome editing in Clostridium
ljungdahlii, an autotrophic gas-fermenting bacterium
He Huang, Changsheng Chai, Ning Li, Peter Rowe, Nigel
Peter Minton, Sheng Yang, Weihong Jiang, and Yang Gu
ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00044 • Publication Date (Web): 08 Jun 2016
Downloaded from http://pubs.acs.org on June 10, 2016

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 free 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 accessible to all
readers and 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.

ACS Synthetic Biology 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 31 ACS Synthetic Biology

1
2
3
4 1 CRISPR/Cas9-based efficient genome editing in Clostridium ljungdahlii, an
5
6 2 autotrophic gas-fermenting bacterium
7
8
9 3 He Huang†1, Changsheng Chai†1, Ning Li1, Pete Rowe2, Nigel P. Minton2, Sheng
10
11
12 4 Yang1, 3, Weihong Jiang1, 3, and Yang Gu*1, 4
13
14 1
15 5 Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology,
16
17 6 Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai
18
19
20 7 200032, China
21
22 8
2
Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre
23
24
25 9 (SBRC), School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD,
26
27 10 UK.
28
29 3
30 11 Jiangsu National Synergetic Innovation Center for Advanced Materials, SICAM, 200
31
32 12 North Zhongshan Road, Nanjing 210009, China
33
34
4
35 13 Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130
36
37 14 Meilong Road, Shanghai 200237, China.
38
39
40 15
41
42
16 Corresponding author
43
44
45 17 *Phone: 86-21-54924178. Fax: 86-21-54924015. E-mail: [email protected].
46
47
18
48
49
50 19 Author Contributions
51
52 †
20 H.H. and C.C. contributed equally to this work. H.H, C.C and N.L. performed the
53
54
55 21 experiments. Y.G., H.H and N.P.M. designed the experiments and wrote the
56
57
22 manuscript. P.R., S.Y. and W.J. assisted in experimental design and data analysis.
58
59
60 1

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 2 of 31

1
2
3
4 23 ABSTRACT:
5
6 24
7
8
9 25 Acetogenic bacteria have the potential to convert single carbon gases (CO and CO2)
10
11 26 into a range of bulk chemicals and fuels. Realization of their full potential is being
12
13
14 27 impeded by the absence of effective genetic tools for high throughput genome
15
16 28 modification. Here we report the development of a highly efficient CRISPR/Cas9
17
18
19 29 system for rapid genome editing of Clostridium ljungdahlii, a paradigm for the
20
21 30 commercial production of ethanol from synthesis gas. Following the experimental
22
23
24 31 selection of two promoters (Pthl and ParaE) for expression of cas9 and the requisite
25
26 32 single guide RNA (sgRNA), the efficiency of system was tested by making precise
27
28
29 33 deletions of four genes, pta, adhE1, ctf and pyrE. Deletion efficiencies were
30
31 34 100%, >75%, 100% and >50%, respectively. The system overcomes the deficiencies
32
33
34 35 of currently available tools (more rapid, no added antibiotic resistance gene, scar-less
35
36 36 and minimal polar effects) and will find utility in other acetogens, including pathogen
37
38
39 37 Clostridium difficile.
40
41
38
42
43
44 39 KEYWORDS: CRISPR/Cas9, rapid genome editing, strong promoters, Clostridium
45
46
40 ljungdahlii, autotrophic bacterium, gas fermentation
47
48
49
50
51
52
53
54
55
56
57
58
59
60 2

ACS Paragon Plus Environment

Page 3 of 31 ACS Synthetic Biology

1
2
3
4 41 The use of fossil fuels is no longer tenable. A finite resource, their use is damaging the
5
6 42 environment through pollution and global warming. Alternative, environmentally
7
8
9 43 friendly sources of chemicals and fuels are required. To date the focus has been on
10
11 44 using lignocellulose as a feedstock for microbial fermentation. However, its
12
13
14 45 recalcitrance to deconstruction is making the development of economic processes
15
16 46 extremely challenging. One alternative is to directly capture carbon before
17
18
19 47 incorporation into lignocellulosic biomass. Acetogenic bacteria, typified by
20
21 48 Clostridium ljungdahlii, are able to capture carbon (CO or CO2) through the
22
23
24 49 Wood-ljungdahlii pathway, allowing them to grow on a spectrum of waste gases from
25
26 50 industry (e.g., steel manufacture and oil refining, coal and natural gas) to produce
27
28
29 51 ethanol (1-3). They can also consume ‘synthesis gas’ (mixtures of CO, CO2 and H2)
30
31 52 made from the gasification of renewable resources, such as biomass in the form of
32
33
34 53 domestic or agricultural waste. Acetogenic gas fermentation can, therefore, produce
35
36 54 ethanol in any geographic region without competing for food or land resources (1-4).
37
38
39 55 The real potential of acetogens, however, resides in their capacity to produce
40
41
56 chemicals and fuels other than ethanol (5). This will require the redesign and
42
43
44 57 implementation of more efficient metabolic pathways, adapting them to high
45
46
58 performing manufacturing processes. The first tentative steps towards such goals have
47
48
49 59 been made in recent years through the development and implementation of classical
50
51
60 tools for the modification of acetogens such as Clostridium ljungdahlii and the closely
52
53
54 61 related Clostridium autoethanogenum (5-9). The tools used to date, however, have
55
56
57
62 been limited to either the introduction of autonomous plasmids encoding selected
58
59
60 3

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 4 of 31

1
2
3
4 63 enzymes involved in product formation (5), the generation of insertional mutants
5
6 64 using ClosTron mutagenesis (6) or the insertion of antibiotic resistance genes by
7
8
9 65 homologous recombination (8) and the single crossover integration of suicide
10
11 66 plasmids (9). In other work, a butyrate production from C. acetobutylicum was
12
13
14 67 integrated into the C. ljungdahlii genome via homologous recombination, and
15
16 68 thereafter modified by excision of the catP antibiotic resistance marker used for
17
18
19 69 selection of the integrated DNA. The latter was achieved through the action of an
20
21 70 introduced heterologous cre recombinase on loxP target sites that flanked the catP
22
23
24 71 gene (7). Despite this progress, the developed methods and tools are in the main
25
26 72 cumbersome and time consuming, being reliant on a number of labor intensive
27
28
29 73 screening steps to identified the desired cell lines. Moreover, their deployment results
30
31 74 in mutants with properties that are less than ideal. Thus, those mutants generate by
32
33
34 75 single crossover integration of plasmids can revert through plasmid excision as a
35
36 76 consequence of recombination between the duplicated regions of homology that
37
38
39 77 mediated plasmid integration (9). On the other hand, the presence of inserted DNA in
40
41
78 those mutants made using the ClosTron (6) or via double crossover integration of
42
43
44 79 mutant alleles carrying antibiotic resistance genes (8), generate mutants that are not
45
46
80 immune from polar effects on downstream genes. Moreover, even in those instances
47
48
49 81 where the selectable marker is removed using Cre-LoxP technology, the final strain
50
51
82 inevitably carries a scar in the chromosome (7).
52
53
54 83 CRISPRs (clustered regularly interspaced short palindromic repeats) function as a
55
56
57
84 prokaryotic acquired immune system, conferring resistance to exogenous genetic
58
59
60 4

ACS Paragon Plus Environment

Page 5 of 31 ACS Synthetic Biology

1
2
3
4 85 elements such as plasmids and phages (10). In type I and III systems, the pre-crRNA
5
6 86 transcript is cleaved within the repeats by CRISPR-associated ribonucleases, releasing
7
8
9 87 multiple small crRNAs. In type II systems, crRNA-tracrRNA hybrids complex with
10
11 88 Cas9 to mediate interference. The CRISPR nuclease Cas9 is targeted by a short guide
12
13
14 89 RNA that recognizes the target DNA via Watson-Crick base pairing. The guide
15
16 90 sequence within these CRISPR RNAs can be easily replaced by a sequence of interest
17
18
19 91 to retarget the Cas9 nuclease to a gene of choice. In recent years, the CRISPR-Cas9
20
21 92 system has been developed into a powerful tool for high-efficiency genome editing
22
23
24 93 (10, 11), by using a Cas9 nuclease, CRISPR RNA (crRNA) and trans-activating
25
26 94 crRNA (tracrRNA), which work together to bind and cut at any target DNA site
27
28
29 95 (12-14), or a simplified system, in which crRNA and tracrRNA were reprogrammed to
30
31 96 yield a chimeric single guide RNA (sgRNA) that was more easily designed and
32
33
34 97 manipulated (12, 15, 16). Recently, this powerful genetic tool has been harnessed for
35
36 98 genome editing in two industrially relevant clostridial species, Clostridium
37
38
39 99 cellulolyticum and Clostridium beijerinckii (17, 18). The former is a cellulolytic
40
41
100 bacterium that is capable of directly converting lignocellulosic biomass into chemical
42
43
44 101 commodities (19, 20), while the latter is highly effective at fermenting
45
46
102 biomass-derived hydrolysates (21-23). Here we sought to establish a similar system in
47
48
49 103 C. ljungdahlii, a paradigm for clostridial acetogens, based on a CRISPR/Cas9-based
50
51
104 editing system developed in our laboratory for use in E.coli (24).
52
53
54 105 As shown in Figures 1A and B, we constructed a plasmid that contained a sgRNA
55
56
57
106 expression cassette and the Streptococcus pyogenes cas9, together with the requisite C.
58
59
60 5

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 6 of 31

1
2
3
4 107 ljungdahlii-derived DNA required to mediate repair of the CRISPR/Cas9 catalyzed
5
6 108 double strand break. For effective operation of the envisaged system, it is crucial that
7
8
9 109 the incorporated CRISPR/Cas9 elements are well expressed in C. ljungdahlii under
10
11 110 the growth conditions to be used. Accordingly, a number of heterologous promoters
12
13
14 111 from C. acetobutylicum were cloned upstream of a promoter-less lacZ reporter gene,
15
16 112 introduced into C. ljungdahlii on an autonomous plasmid, and the relative production
17
18
19 113 of β-galactosidase was assayed at 24, 48 and 72 h time points during heterotrophic
20
21 114 growth on fructose as the carbon source (Figure 2). Promoters from C. ljungdahlii
22
23
24 115 were deliberately avoided to rule out unintended homologous recombination between
25
26 116 the final plasmid and the chromosome. The promoters tested were the ParaE promoter
27
28
29 117 of the C. acetobutylicum gene CA_C1339, together with the promoters of several
30
31 118 solventogenic genes that had previously been characterized, namely, Pthl (25, 26), Pptb
32
33
34 119 (25, 26), and Padc (26). The data obtained demonstrated that Pthl and ParaE promoters
35
36 120 exhibited much greater activity at all three time points than Pptb and Padc. The Pthl and
37
38
39 121 ParaE promoters were, therefore, selected to mediate expression of Cas9 and sgRNA,
40
41
122 respectively, in C. ljungdahlii.
42
43
44 123 Having established the basic components of the C. ljungdahlii CRISPR/Cas9
45
46
124 system, a plasmid (pMTLcas-pta, Figure 1A) capable of targeting the pta
47
48
49 125 (CLJU_c12770) gene encoding phosphotransacetylase was assembled and transferred
50
51
126 into C. ljungdahlii by electroporation. In parallel, two control plasmids, pMTLcasc
52
53
54 127 (lacking the pta homology arms) and pMTLHa (carrying the pta homology arms, but
55
56
57
128 lacking Cas9 and sgRNA), were also electroporated into C. ljungdahlii (Table S2).
58
59
60 6

ACS Paragon Plus Environment

Page 7 of 31 ACS Synthetic Biology

1
2
3
4 129 Transformed cells were plated on YTF (27) solidified media supplemented with 5
5
6 130 µg/mL thiamphenicol and those colonies visible after 3 days of incubation subjected
7
8
9 131 to PCR screening in order to detect the desired double-crossover genotype. The
10
11 132 primers were designed such that a 3,313-bp PCR amplified fragment would be
12
13
14 133 generated from the wild-type pta allele, whereas the mutant pta allele would generate
15
16 134 a 2,300-bp DNA fragment. Of the 8 and 10 colonies obtained in two independent
17
18
19 135 experiments (Table 1) evidence for the presence of the desired pta deletion was
20
21 136 obtained in all cases. In contrast, no pta-deletion events were observed in the
22
23
24 137 electro-transformants that carried the control plasmids, indicating that the generation
25
26 138 of mutants was specific to the presence of a fully functional CRISPR/Cas9 system. In
27
28
29 139 a significant number of cases, however, the PCR screening suggested that the colonies
30
31 140 were composed of both mutant and wild-type populations, as evidenced by the
32
33
34 141 presence of both the 2,300-bp and 3,313-bp PCR- amplified DNA fragments (Table 1
35
36 142 and Figure 1C). These data may suggest that, at this stage in the development of the
37
38
39 143 system and protocol, that complete cleavage of the target site by the Cas9/sgRNA
40
41
144 complex is not occurring. More importantly, however, several colonies represented
42
43
44 145 pure, clonal populations of the desired mutant, as evidenced by the generation of only
45
46
146 a 2,300-bp PCR-amplified DNA fragment, and the complete absence of the 3,313-bp
47
48
49 147 DNA fragment associated with the wild-type genotype (Figure 1C). The 2,300-bp
50
51
148 PCR-amplified DNA fragment was also extracted and sequenced to further confirm
52
53
54 149 the desired deletion (Fig. S1 in supplementary data). These data show the desired
55
56
57
150 deletion mutant can be isolated in a single step, requiring no further restreaking and
58
59
60 7

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 8 of 31

1
2
3
4 151 screening to identify pure clonal sub-populations of mutants.
5
6 152 Phenotypic characterization of a pta mutant revealed that the production of acetic
7
8
9 153 acid had been substantially reduced compared to the wild-type strain (Figure 3). This
10
11 154 is consistent with the central role of phosphotransacetylase in the acetate acid-forming
12
13
14 155 pathway (28). It was also notable that the growth rate of the mutants was significantly
15
16 156 impaired in comparison to the wild-type (Figure 3). The pta-dependent acetic
17
18
19 157 acid-forming pathway represents a major ATP donor in C. ljungdahlii during syngas
20
21 158 fermentation (5). Impairment of growth is, therefore, most likely a consequence of the
22
23
24 159 reduction in the cells ability to generate ATP.
25
26 160 To further exemplify the system, three further C. ljungdahlii genes were targeted.
27
28
29 161 These were, adhE1 (CLJU_c16510, encoding a bifunctional aldehyde/alcohol
30
31 162 dehydrogenase), ctf (CLJU_c39430, which encodes acyl-CoA transferase) and pyrE
32
33
34 163 (CLJU_c35680, which encodes orotate phosphoribosyltransferase). In every instance,
35
36 164 evidence for the presence of mutant populations in some, or all, of the transformant
37
38
39 165 colonies was obtained by PCR screening. In contrast to the result with pta, some of
40
41
166 the colonies, although a minority, appeared to be composed of entirely wild-type cells.
42
43
44 167 Nevertheless, the majority of the colonies were composed of either mutant or a
45
46
168 mixture of mutant and wild-type populations. In the case of ctf, all (21 in total) of the
47
48
49 169 colonies screened were composed of mutant and wild-type cells. In the case of pyrE,
50
51
170 the majority (15 out of 22) comprised mutant and wild-type cells, with one pure
52
53
54 171 mutant and 6 wild-type colonies. In contrast, in the case of adhE1, no mixed colonies
55
56
57
172 were evident. Rather the transformant colonies comprised 14 deletion mutants and 4
58
59
60 8

ACS Paragon Plus Environment

Page 9 of 31 ACS Synthetic Biology

1
2
3
4 173 wild-type cell lines.
5
6 174 To address the issue of mixed populations, colonies carrying both mutant and
7
8
9 175 wild-type cells from the ctf and pyrE deletion experiments were resuspended in 20 µL
10
11 176 liquid YTF medium (supplemented with 5 µg/mL thiamphenicol) and re-streaked onto
12
13
14 177 YTF agar plates (supplemented with 5 µg/mL thiamphenicol). Following two days of
15
16 178 incubation, 15 well isolated individual colonies from either the ctf or pyrE mixed
17
18
19 179 mutant cell lines, respectively, were selected for PCR screening. In all cases, a single
20
21 180 PCR-amplified DNA fragment was obtained consistent with the mutant allele (Table
22
23
24 181 2). These data indicate that, at least in these instances, pure mutant populations can
25
26 182 easily be isolated from primary colonies composed of mixed mutant and wild-type
27
28
29 183 alleles by one-step subculturing. This may suggest that the prolonged incubation of
30
31 184 cells in the continued presence of the active, targeted Cas9/sgRNA plasmid leads to
32
33
34 185 total conversion of cells to mutants. Alternatively, ctf and pyrE mutants may possess a
35
36 186 growth advantage over the wild-type cells, leading to their predominance as well
37
38
39 187 isolated colonies. All these three deletion events were further confirmed by
40
41
188 sequencing (Fig. S1 in supplementary data)
42
43
44 189 Phenotypic characterization of an adhE1 mutant revealed that the production of
45
46
190 ethanol had been significantly reduced compared to the wild-type strain (Figure 4A).
47
48
49 191 This is consistent with the essential role of bifunctional aldehyde/alcohol
50
51
192 dehydrogenase in the ethanol-forming pathway (28). The ctf mutant M-ctf also
52
53
54 193 showed impaired growth compared to the wild-type strain, leading to decreased
55
56
57
194 production of acetic acid and ethanol (Figure 4B). The phenotypic effect of deleting
58
59
60 9

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 10 of 31

1
2
3
4 195 ctf is in agreement with the previously reported result, in which ctf was inactivate by
5
6 196 single-crossover homologous recombination (7). In addition, the deletion of pyrE
7
8
9 197 resulted in the desired FOA-resistant cells, which can grow on solidified medium
10
11 198 supplemented with FOA (Figure 4C).
12
13
14 199 In summary, here we report the first demonstration of CRISPR/Cas9 genome
15
16 200 editing in an industrially important clostridial acetogen, C. ljungdahlii. This genetic
17
18
19 201 tool facilitates rapid and precise chromosomal manipulation in C. ljungdahlii and
20
21 202 overcomes intrinsic limitations in those methods described to date for constructing
22
23
24 203 mutant strains for industrial applications. Mutants made are scar-less and devoid of
25
26 204 undesirable antibiotic resistance markers, an essential feature of chassis destined for
27
28
29 205 process scale-up. The system should prove applicable to other autotrophic Clostridium
30
31 206 species for efficient genome editing, both industrially important (e.g., C.
32
33
34 207 autoethanogenum) and pathogens (e.g., Clostridium difficile). More importantly, our
35
36 208 systems will form the basis of more elaborate implementations of Synthetic Biology
37
38
39 209 approaches in acetogens, such as gene regulation, elucidating gene function, pathway
40
41
210 engineering and more sophisticated cell factory designs.
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 10

ACS Paragon Plus Environment

Page 11 of 31 ACS Synthetic Biology

1
2
3
4 211 METHODS
5
6 212 Strains, Media, and Reagents
7
8
9 213 The E.coli host strain DH5α E. coli strain was used for plasmid cloning and
10
11 214 maintenance. It was grown in LB medium supplemented with chloramphenicol (12.5
12
13
14 215 µg/mL) when needed. The C. ljungdahlii strain used was DSM 13528. It was grown
15
16 216 in YTF medium (27) or a modified ATCC medium 1754 (2-fold increased
17
18
19 217 concentration of all the mineral elements; fructose was removed) with a headspace of
20
21 218 CO-CO2-H2-N2 (56%/20%/9%/15%; pressurized to 0.2 MPa). In addition, 5 µg/mL of
22
23
24 219 thiamphenicol (Wako Pure Chemical Industries, Osaka, Japan) was supplemented as
25
26 220 needed for plasmid selection. KOD plus Neo and KOD FX DNA polymerase (Toyobo,
27
28
29 221 Osaka, Japan) was used for high fidelity DNA amplification and PCR screening of the
30
31 222 desired genotypes of C. ljungdahlii. The oligonucleotide primers used were
32
33
34 223 synthesized by GenScript (Nanjing, China). Plasmids were constructed using
35
36 224 conventional restriction digestion enzymes and ligase purchased from Thermo Fisher
37
38
39 225 Scientific (USA）and Takara (Dalian, China), respectively, or assembled through the
40
41
226 ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd, Nanjing,
42
43
44 227 China). Plasmid isolation and DNA purification were performed with kits purchased
45
46
228 from Axygen (Hangzhou, China).
47
48
49 229
50
51
52
230 Plasmid Construction
53
54 231 The CRISPR/Cas9 editing plasmid pMTLcas-pta was assembled from the
55
56
57
232 following fragments: linear plasmid pMTL83151 (29) obtained by double digestion
58
59
60 11

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 12 of 31

1
2
3
4 233 with SacI and XhoI; the native cas9 of Streptococcus pyogenes was obtained by PCR
5
6 234 using the primers cas9fw/cas9rw and pCas as the template; the Pthl and ParoE
7
8
9 235 promoters were obtained from Clostridium acetobutylicum using the primers
10
11 236 pthlrw/pthlfw and paraEfw/paraErw, respectively; sgRNA (which targets the 20-nt
12
13
14 237 target spacer of the pta gene) was obtained by PCR using the primers
15
16 238 ptagRNAs/gRNAaster and pTargetF as template; the two homologous arms (HAs)
17
18
19 239 that flank the open reading frame of pta were amplified from C. acetobutylicum
20
21 240 genomic DNA using the primers ptauas/ptauas and ptads/ptadas.
22
23
24 241 Here, every 20-nt target sequence (5'-N20NGG-3', where N represents any
25
26 242 nucleotide) chosen for sgRNA targeting was examined in advance by alignment
27
28
29 243 search (NCBI BLAST), aiming to ensure no sequence with high nucleotide acid
30
31 244 sequence similarity in the genome and thus avoid the potential “off-target” in gene
32
33
34 245 deletion. Then, the sgRNA scaffold and ParaE promoter were assembled by an overlap
35
36 246 extension PCR, which resulted in the ParaE-sgRNA fragment. This fragment was then
37
38
39 247 assembled with the above HAs fragments by a second round of overlap extension
40
41
248 PCR, which yielded the fragment ParaE-sgRNA-HA. Thereafter, the linear
42
43
44 249 pMTL83151 vector, and DNA fragments encompassing Pthl, cas9, and
45
46
250 ParaE-sgRNA-HA were assembled to give the CRISPR/Cas9 editing plasmid
47
48
49 251 pMTLcas-pta using the ClonExpress MultiS One Step Cloning Kit (Fig. 1A). When
50
51
252 preparing the other three plasmids for the adhE1, ctf and pyrE deletion, the
52
53
54 253 pMTLcas-pta plasmid was used as the template, and the sgRNA and homologous
55
56
57
254 arms cassette were changed accordingly.
58
59
60 12

ACS Paragon Plus Environment

Page 13 of 31 ACS Synthetic Biology

1
2
3
4 255 To construct the pMTLcasc control plasmid (cas9+sgRNA, without HAs), the
5
6 256 pMTLcas-pta plasmid was digested with XbaI and XhoI to remove the original
7
8
9 257 sgRNA and HAs. The resulting linear vector was then assembled (using the
10
11 258 ClonExpress MultiS One Step Cloning Kit) with a single pta-specific sgRNA
12
13
14 259 fragment that was PCR-amplified from the pTargetF template using the primers
15
16 260 ptagRNAs/gRNAasre. Similarly, the other control plasmid pMTLHa (HAs, without
17
18
19 261 cas9 and sgRNA) was obtained by removing cas9 and sgRNA from pMTLcas-pta by
20
21 262 NotI-SalI double digestion, followed by blunt-ending and self-ligation.
22
23
24 263 The reporter plasmids were constructed as follows: the chloramphenicol
25
26 264 acetyltransferase gene (catP) was amplified from the pMTL83151 plasmid with the
27
28
29 265 catP-SacI-For/catP-ClaI-Rev primers and digested with SacI/ClaI. The cleaved
30
31 266 fragment was then ligated with pXY1 plasmid digested with the same restriction
32
33
34 267 enzymes, to yield the plasmid pLXY1. Next, the lacZ reporter gene was excised from
35
36 268 pIMP1-Pthl-LacZ by BamHI-SmaI digestion and ligated with the pLXY1 plasmid
37
38
39 269 digested with the same restriction enzymes, to give the plasmid pLXY1-Pthl-LacZ.
40
41
270 Subsequently, the Pptb, Padc, ParaE promoters were PCR-amplified using genomic DNA
42
43
44 271 of C. acetobutylicum ATCC 824 as template and Pptb-PstI-For/Pptb-BamHI-Rev,
45
46
272 Padc-PstI-For/Padc-BamHI-Rev, and ParaE-PstI-For/ParaE-BamHI-Rev as primers.
47
48
49 273 This reaction was digested with PstI/BamHI and ligated into the pLXY1-Pthl-LacZ
50
51
274 plasmid, which was also digested with the same restriction enzymes to replace the
52
53
54 275 original Pthl promoter. The outcomes of this procedure were three new reporter
55
56
57
276 plasmids, namely pLXY1-Pptb-LacZ, pLXY1-Padc-LacZ and pLXY1-ParaE-LacZ.
58
59
60 13

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 14 of 31

1
2
3
4 277
5
6 278 Gene Deletion by CRISPR/Cas9 Plasmids
7
8
9 279 C. ljungdahlii was grown anaerobically at 37°C. The gene deletion was achieved by
10
11 280 delivering the CRISPR/Cas9 editing plasmid into C. ljungdahlii by electroporation.
12
13
14 281 Electrocompetent cells were prepared according to the previously described protocol
15
16 282 (8). The electroporation procedure was slightly modified. Briefly, 200 µL of
17
18
19 283 electrocompetent cells were thawed on ice and then mixed with 4 µg of plasmid DNA
20
21 284 recovered from E. coli DH5α for approximately 10 min. The mixture of
22
23
24 285 electrocompetent cells and plasmid DNA was then transferred into a 2 mm-gap
25
26 286 electroporation cuvette (Harvard Apparatus, MA, USA) and pulsed with parameters
27
28
29 287 set at 1.0 kV, 200 Ω and 50 µF using Gene Pulser Xcell microbial electroporation
30
31 288 system (Bio-Rad). The cells were recovered in 2 mL YTF medium supplemented with
32
33
34 289 0.4% L-cysteine for 6 h. Then, they were plated onto YTF agar plates (supplemented
35
36 290 with 5 µg/mL of thiamphenicol) at 37°C for approximately three days. When the
37
38
39 291 colonies were visible on the agar plate, the primers listed in the Supplementary Table
40
41
292 S2 were used for the PCR screening of the desired deletion events. Then, the
42
43
44 293 PCR-amplified DNA fragments were extracted and sequenced to further confirm the
45
46
294 deletions.
47
48
49 295
50
51
52
296 β-Galactosidase Assays
53
54 297 The plasmids pLXY1-Pthl-LacZ, pLXY1-Pptb-LacZ, pLXY1-Padc-LacZ, and
55
56
57
298 pLXY1-ParaE-LacZ were transferred into C. ljungdahlii by electro-transformation. C.
58
59
60 14

ACS Paragon Plus Environment

Page 15 of 31 ACS Synthetic Biology

1
2
3
4 299 ljungdahlii cells that harbored the reporter plasmids were grown anaerobically at
5
6 300 37°C and harvested at 24, 48 and 72 h by centrifugation at 12, 000 g for 5 min. The
7
8
9 301 cell pellets were dissolved in the B-PER reagent (Thermo Scientific Pierce®, USA)
10
11 302 and vortexed for 1 min for lysing the cells. The resulting cell lysate was heat-treated
12
13
14 303 at 60°C for 30 min to remove heat-unstable proteins and, then, centrifuged at 12, 000
15
16 304 g for 30 min. The supernatant was used for the β-galactosidase assays as previously
17
18
19 305 reported (25).
20
21 306
22
23 307 Phenotypic Identification of Cells with pyrE Deletion
24
25 308 Cells with pyrE deletion can be identified on growth medium supplemented with
26
27
28 309 5-fluoroorotic acid (FOA), as FOA is highly toxic to cells harboring pyrE-based
29
30 310 pyrimidine synthetic pathway. The assay was performed as previously described (30),
31
32
33 311 excepted that solidified YTF medium supplemented with 2 mg/mL FOA was used as
34
35 312 the assay medium.
36
37
38 313
39
40 314 Fermentation
41
42
43 315 All the C. ljungdahlii mutants were serially subcultured in YTF liquid medium (no
44
45 316 antibiotic added) to eliminate the plasmid before fermentation. The fermentation was
46
47
48 317 performed in 125 mL Wheaton serum bottles (Sigma-Aldrich, USA) with 30 mL
49
50 318 working volume. Briefly, 100 µL of frozen stock was inoculated into 5 mL YTF
51
52
53 319 liquid medium and incubated anaerobically at 37°C for 24 h. When the optical density
54
55 320 (OD600) of cells reached 0.5-1.0, approximately 5% (vol/vol) of the inoculum was
56
57
58 321 transferred into 30 mL of a modified ATCC medium 1754 (2-fold increased
59
60 15

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 16 of 31

1
2
3
4 322 concentration of all the mineral elements; fructose was removed) with a headspace of
5
6 323 CO-CO2-H2-N2 (56%/20%/9%/15%; pressurized to 0.2 MPa) for fermentation. The
7
8
9 324 bottles were incubated horizontally in a shaking incubator at 37°C and 150 rpm. The
10
11 325 culture was sampled every 24 h and, then, the gases were added into the headspace
12
13
14 326 (again at 0.2 MPa).
15
16 327 Analytical Methods
17
18
19 328 Cell growth was followed by measuring the absorbance of the sample at A600
20
21 329 (OD600) using a spectrophotometer (U-1800, Hitachi, Japan). The concentrations of
22
23
24 330 ethanol and acetate were measured by an internal standard method using gas
25
26 331 chromatography (7890 A, Agilent, Wilmington, DE, USA) equipped with a flame
27
28
29 332 ionization detector and a capillary column (Alltech ECTM-WAX). The analysis was
30
31 333 carried out under the following conditions: oven temperature, programmed from 85 to
32
33
34 334 150°C; injector temperature, 250°C; detector temperature, 300°C; nitrogen (carrier
35
36 335 gas) flow rate, 20 mL/min; hydrogen flow rate, 30 mL/min; air flow rate, 400 mL/min.
37
38
39 336 The internal standards were isobutanol, isobutyric acid and hydrochloric acid (for
40
41
337 acidification).
42
43
44 338
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 16

ACS Paragon Plus Environment

Page 17 of 31 ACS Synthetic Biology

1
2
3
4 339 ACKNOWLEDGEMENTS
5
6 340 This work was funded by the National High-tech Research and Development
7
8
9 341 Program of China (2015AA020202), the Chinese Academy of Sciences
10
11 342 (KSZD-EW-Z-017-2 and KGZD-EW-606), the Synthetic Biology China-UK
12
13
14 343 Partnering Award (Utilizing Steel Mill 'Off-Gas' for Chemical Commodity Production
15
16 344 using Synthetic Biology), funded by the BBSRC (BB/L01081X/1) and the Chinese
17
18
19 345 Academy of Sciences, the National Natural Science Foundation of China (31370133
20
21 346 and 31121001) and Youth Innovation Promotion Association CAS.
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 17

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 18 of 31

1
2
3
4 347 SUPPORTING INFORMATION
5
6 348 Table S1 Strains and plasmids in this study
7
8
9 349 Table S2 Oligonucleotides used in this study
10
11 350 Fig. S1 Confirmation of the deletion of pta, adhE1, ctf and pyrE by
12
13
14 351 sequencing
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 18

ACS Paragon Plus Environment

Page 19 of 31 ACS Synthetic Biology

1
2
3
4 352 References
5
6 353 1. Hawkins, A. S., McTernan, P. M., Lian, H., Kelly, R. M., and Adams, M. W.
7
8
9 354 (2013) Biological conversion of carbon dioxide and hydrogen into liquid fuels
10
11 355 and industrial chemicals. Curr. Opin. Biotechnol. 24, 376-384.
12
13
14 356 2. Wilkins, M. R., and Atiyeh, H. K. (2011) Microbial production of ethanol from
15
16 357 carbon monoxide. Curr. Opin. Biotechnol. 22, 326-330.
17
18
19 358 3. Köpke, M., Mihalcea, C., Bromley, J. C., and Simpson, S. D. (2011)
20
21 359 Fermentative production of ethanol from carbon monoxide. Curr. Opin.
22
23
24 360 Biotechnol. 22, 320-325.
25
26 361 4. Tracy, B. P., Jones, S. W., Fast, A. G., Indurthi, D. C., and Papoutsakis, E. T.
27
28
29 362 (2012) Clostridia: the importance of their exceptional substrate and metabolite
30
31 363 diversity for biofuel and biorefinery applications. Curr. Opin. Biotechnol. 23,
32
33
34 364 364-381.
35
36 365 5. Köpke, M., Held, C., Hujer, S., Liesegang, H., Wiezer, A., Wollherr, A.,
37
38
39 366 Ehrenreich, A., Liebl, W., Gottschalk, G., and Dürre, P. (2010) Clostridium
40
41
367 ljungdahlii represents a microbial production platform based on syngas. Proc.
42
43
44 368 Natl. Acad. Sci. U. S. A. 107, 13087-13092.
45
46
369 6. Mock, J., Zheng, Y., Mueller, A. P., Ly, S., Tran, L., Segovia, S., Nagaraju, S.,
47
48
49 370 Kopke, M., Dürre, P., and Thauer, R. K. (2015) Energy conservation
50
51
371 associated with ethanol formation from H2 and CO2 in Clostridium
52
53
54 372 autoethanogenum involving electron bifurcation. J. Bacteriol. 197, 2965-2980.
55
56
57
373 7. Ueki, T., Nevin, K. P., Woodard, T. L., and Lovley, D. R. (2014) Converting
58
59
60 19

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 20 of 31

1
2
3
4 374 carbon dioxide to butyrate with an engineered strain of Clostridium ljungdahlii.
5
6 375 mBio 5, e01636-01614.
7
8
9 376 8. Leang, C., Ueki, T., Nevin, K. P., and Lovley, D. R. (2013) A genetic system
10
11 377 for Clostridium ljungdahlii: a chassis for autotrophic production of
12
13
14 378 biocommodities and a model homoacetogen. Appl. Environ. Microbiol. 79,
15
16 379 1102-1109.
17
18
19 380 9. Tremblay, P. L., Zhang, T., Dar, S. A., Leang, C., and Lovley, D. R. (2012) The
20
21 381 Rnf complex of Clostridium ljungdahlii is a proton-translocating
22
23
24 382 ferredoxin:NAD+ oxidoreductase essential for autotrophic growth. mBio 4,
25
26 383 e00406-00412.
27
28
29 384 10. Copeland, M. F., Politz, M. C., and Pfleger, B. F. (2014) Application of TALEs,
30
31 385 CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes. Curr. Opin.
32
33
34 386 Biotechnol. 29, 46-54.
35
36 387 11. Fineran, P. C., and Dy, R. L. (2014) Gene regulation by engineered
37
38
39 388 CRISPR-Cas systems. Curr. Opin. Microbiol. 18, 83-89.
40
41
389 12. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and
42
43
44 390 Charpentier, E. (2012) A programmable dual-RNA-guided DNA endonuclease
45
46
391 in adaptive bacterial immunity. Science 337, 816-821.
47
48
49 392 13. Jiang, W., Bikard, D., Cox, D., Zhang, F., and Marraffini, L. A. (2013)
50
51
393 RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat.
52
53
54 394 Biotechnol. 31, 233-239.
55
56
57
395 14. Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada,
58
59
60 20

ACS Paragon Plus Environment

Page 21 of 31 ACS Synthetic Biology

1
2
3
4 396 Z. A., Eckert, M. R., Vogel, J., and Charpentier, E. (2011) CRISPR RNA
5
6 397 maturation by trans-encoded small RNA and host factor RNase III. Nature 471,
7
8
9 398 602-607.
10
11 399 15. DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., and Church, G. M.
12
13
14 400 (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas
15
16 401 systems. Nucleic Acids Res. 41, 4336-4343.
17
18
19 402 16. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J.
20
21 403 E., and Church, G. M. (2013) RNA-guided human genome engineering via
22
23
24 404 Cas9. Science 339, 823-826.
25
26 405 17. Wang, Y., Zhang, Z. T., Seo, S. O., Choi, K., Lu, T., Jin, Y. S., and Blaschek, H.
27
28
29 406 P. (2015) Markerless chromosomal gene deletion in Clostridium beijerinckii
30
31 407 using CRISPR/Cas9 system. J. Biotechnol. 200, 1-5.
32
33
34 408 18. Xu, T., Li, Y., Shi, Z., Hemme, C. L., Li, Y., Zhu, Y., Van Nostrand, J. D., He,
35
36 409 Z., and Zhou, J. (2015) Efficient genome editing in Clostridium cellulolyticum
37
38
39 410 via CRISPR-Cas9 nickase. Appl. Environ. Microbiol. 81, 4423-4431.
40
41
411 19. Higashide, W., Li, Y., Yang, Y., and Liao, J. C. (2011) Metabolic engineering
42
43
44 412 of Clostridium cellulolyticum for production of isobutanol from cellulose.
45
46
413 Appl. Environ. Microbiol. 77, 2727-2733.
47
48
49 414 20. Li, Y., Tschaplinski, T. J., Engle, N. L., Hamilton, C. Y., Rodriguez, M., Jr.,
50
51
415 Liao, J. C., Schadt, C. W., Guss, A. M., Yang, Y., and Graham, D. E. (2012)
52
53
54 416 Combined inactivation of the Clostridium cellulolyticum lactate and malate
55
56
57
417 dehydrogenase genes substantially increases ethanol yield from cellulose and
58
59
60 21

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 22 of 31

1
2
3
4 418 switchgrass fermentations. Biotechnol. Biofuels 5, 2.
5
6 419 21. Xiao, H., Li, Z., Jiang, Y., Yang, Y., Jiang, W., Gu, Y., and Yang, S. (2012)
7
8
9 420 Metabolic engineering of D-xylose pathway in Clostridium beijerinckii to
10
11 421 optimize solvent production from xylose mother liquid. Metab. Eng. 14,
12
13
14 422 569-578.
15
16 423 22. Wang, Y., and Blaschek, H. P. (2011) Optimization of butanol production from
17
18
19 424 tropical maize stalk juice by fermentation with Clostridium beijerinckii
20
21 425 NCIMB 8052. Bioresour. Technol. 102, 9985-9990.
22
23
24 426 23. Du, T. F., He, A. Y., Wu, H., Chen, J. N., Kong, X. P., Liu, J. L., Jiang, M., and
25
26 427 Ouyang, P. K. (2013) Butanol production from acid hydrolyzed corn fiber with
27
28
29 428 Clostridium beijerinckii mutant. Bioresour. Technol. 135, 254-261.
30
31 429 24. Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., and Yang, S. (2015) Multigene
32
33
34 430 editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl.
35
36 431 Environ. Microbiol. 81, 2506-2514.
37
38
39 432 25. Girbal, L., Mortier-Barriere, I., Raynaud, F., Rouanet, C., Croux, C., and
40
41
433 Soucaille, P. (2003) Development of a sensitive gene expression reporter
42
43
44 434 system and an inducible promoter-repressor system for Clostridium
45
46
435 acetobutylicum. Appl. Environ. Microbiol. 69, 4985-4988.
47
48
49 436 26. Tummala, S. B., Welker, N. E., and Papoutsakis, E. T. (1999) Development
50
51
437 and characterization of a gene expression reporter system for Clostridium
52
53
54 438 acetobutylicum ATCC 824. Appl. Environ. Microbiol. 65, 3793-3799.
55
56
57
439 27. Humphreys, C. M., McLean, S., Schatschneider, S., Millat, T., Henstra, A. M.,
58
59
60 22

ACS Paragon Plus Environment

Page 23 of 31 ACS Synthetic Biology

1
2
3
4 440 Annan, F. J., Breitkopf, R., Pander, B., Piatek, P., Rowe, P., Wichlacz, A. T.,
5
6 441 Woods, C., Norman, R., Blom, J., Goesman, A., Hodgman, C., Barrett, D.,
7
8
9 442 Thomas, N. R., Winzer, K., and Minton, N. P. (2015) Whole genome sequence
10
11 443 and manual annotation of Clostridium autoethanogenum, an industrially
12
13
14 444 relevant bacterium. BMC genomics 16, 1085.
15
16 445 28. Nolling, J., Breton, G., Omelchenko, M. V., Makarova, K. S., Zeng, Q., Gibson,
17
18
19 446 R., Lee, H. M., Dubois, J., Qiu, D., Hitti, J., Wolf, Y. I., Tatusov, R. L., Sabathe,
20
21 447 F., Doucette-Stamm, L., Soucaille, P., Daly, M. J., Bennett, G. N., Koonin, E.
22
23
24 448 V., and Smith, D. R. (2001) Genome sequence and comparative analysis of the
25
26 449 solvent-producing bacterium Clostridium acetobutylicum. J. Bacteriol. 183,
27
28
29 450 4823-4838.
30
31 451 29. Heap, J. T., Pennington, O. J., Cartman, S. T., and Minton, N. P. (2009) A
32
33
34 452 modular system for Clostridium shuttle plasmids. J. Microbiol. Methods 78,
35
36 453 79-85.
37
38
39 454 30. Heap, J. T., Ehsaan, M., Cooksley, C. M., Ng, Y. K., Cartman, S. T., Winzer,
40
41
455 K., and Minton, N. P. (2012) Integration of DNA into bacterial chromosomes
42
43
44 456 from plasmids without a counter-selection marker. Nucleic Acids Res. 40, e59.
45
46 457
47
48
49
50
51
52
53
54
55
56
57
58
59
60 23

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 24 of 31

1
2
3 458 Table 1. Results of the pta deletion in C. ljungdahlii
4
5 Plasmids Elements Experimental rounds Results Efficiency#
6 (M/P/W/T)*
7
pMTLcasc Cas9+sgRNA — N —
8
9 pMTLHa Homologous arms — N —
10 pMTLcas-pta Cas9+sgRNA+ 1 7/1/0/8 100%
11 Homologous arms
12
13 2 4/6/0/10 100%
14
15
459 *M: number of colonies that harbored mixed wild-type and gene-deleted cells; P:
16
17 460 number of colonies that harbored pure gene-deleted cells; W: number of colonies that
18
19
20 461 harbored pure wild-type cells; T: total number of colonies used for PCR screening.
21
#
22 462 Efficiency: probability of deletion events occurring, calculated as (M+P)/T×100%.
23
24
25 463
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 24

ACS Paragon Plus Environment

Page 25 of 31 ACS Synthetic Biology

1
2
3 464 Table 2. Results of the adhE1, ctf and pyrE deletion in C. ljungdahlii
4 465
5
6 Gene Deletion Experimental Results
7 targets size (bp) rounds Before Efficiency# After Efficiency#
8 subculturing subculturing
9
(M/P/W/T)* (M/P/T)
10
11 adhE1 2,600 1 0/3/1/4 75% — —
12 2 0/11/3/14 78% — —
13 ctf 1,200 1 11/0/0/11 100% 0/5/5 100%
14
2 10/0/0/10 100% 0/10/10 100%
15
16 pyrE 570 1 6/0/6/12 50% 0/7/7 100%
17 2 9/1/0/10 100% 0/8/8 100%
18
19 466 *M: number of colonies that harbored mixed wild-type and gene-deleted cells; P:
20
21
22
467 number of colonies that harbored pure gene-deleted cells; W: number of colonies that
23
24 468 harbored pure wild-type cells; T: total number of colonies used for PCR screening.
25
26 #
27
469 Efficiency: probability of deletion events occurring, calculated as (M+P)/T×100%.
28
29 470
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 25

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 26 of 31

1
2
3
4 471 Fig. 1. Design of the CRISPR/Cas9 system for deletion of the C. ljungdahlii pta gene.
5
6 472 (A) Strategy for the construction of the plasmid pMTLcas-pta. (B) Strategy for pta
7
8
9 473 deletion using pMTLcas-pta. The yellow star indicates the recognition site of the
10
11 474 Cas9-sgRNA complex in the coding region of pta. (C) PCR screening of the pta
12
13
14 475 deletion. The 3.3 and 2.3 kbp bands represent the wild-type and the pta deletion
15
16 476 genotype, respectively. M: marker; WT: wild-type strain.
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38 477
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 26

ACS Paragon Plus Environment

Page 27 of 31 ACS Synthetic Biology

1
2
3
4 478 Fig. 2. Comparison of the strength of the four heterologous promoters (Pthl, ParaE, Pptb
5
6 479 and Padc from C. acetobutylicum ATCC 824) in C. ljungdahlii. Pcontrol: no
7
8
9 480 promoter to drive lacZ expression. Data are representative of three replicates.
10
11 481
12
13 1600 Pthl
thl ParaE
araE
14
15 Pptb
ptb Padc
adc
16
1400 Pcontrol
control
17
18 1200
19
20 1000
U/mg

21
22 800
23
24 600
25
26 400
27
28
29
200
30
31 0
32
33
24 48 72
34 Time (h)
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 27

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 28 of 31

1
2
3
4 482 Fig. 3. Phenotypic effect after the pta deletion in C. ljungdahlii. WT: wild-type strain;
5
6 483 M-pta: pta-deleted mutant. Data are representative of triplicate cultures.
7
8
9 484
Growth Acetate
10 3.0 5.0
11
12 2.5
13 4.0

Concentration(g/L)
Growth (OD 600)

14 2.0
15 WT 3.0 WT
16 1.5
17 M-pta M-pta
2.0
18 1.0
19
20 1.0
0.5
21
22 0.0
0.0
23
24 0 24 48 72 96 0 24 48 72 96
25 Time (h) Time (h)
26
27
Ethanol
28 1.2
29
30 1.0
Concentration (g/L)

31
32 0.8
33 WT
34 0.6
35 M-pta
36
0.4
37
38
39 0.2
40
41 0.0
42 0 24 48 72 96
43
Time (h)
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60 28

ACS Paragon Plus Environment

Page 29 of 31 ACS Synthetic Biology

1
2
3
4 485 Fig. 4. Phenotypic effect after deletion of adhE1 (A), ctf (B) and pyrE (C) in C.
5
6 486 ljungdahlii. WT: wild-type strain; M-adhE1: adhE1-deleted mutant; M-pyrE:
7
8
9 487 pyrE-deleted mutant. Data from (A) and (B) are representative of triplicate cultures.
10
11 488 (A)
12
13 Growth Acetate Ethanol
3.0 5.0 1.2
14
15 1.0

Concentration (g/L)

Concentration (g/L)
2.5 4.0
16
Growth (OD 600)

2.0 0.8
17 3.0
18 1.5 0.6
19 2.0
1.0 0.4
20 1.0
0.5 WT WT 0.2 WT
21 M-adhE1
M-adhE1 M-adhE1
22 0.0 0.0 0.0
23 0 24 48 72 96 0 24 48 72 96 0 24 48 72 96
24 Time (h) Time (h) Time (h)
25
26
27
28 489 (B)
29
30
31 3.0 Growth 5.0 Acetate
32
2.5 4.0
Concentration(g/L)

33
Growth (OD 600)

34 2.0
35 3.0
36 1.5
2.0
37 1.0
38 WT WT
M-ctf 1.0 M-ctf
39 0.5
40
0.0 0.0
41
0 24 48 72 96 120 144 168 192 0 24 48 72 96 120 144 168 192
42
Time (h) Time(h)
43
44
45 1.2 Ethanol
46
47 1.0
Concentration (g/L)

48
0.8
49
50 0.6
51
52 0.4
WT
53 M-ctf
0.2
54
55 0.0
56 0 24 48 72 96 120 144 168 192
57 Time (h)
58
59
60 29

ACS Paragon Plus Environment

 ACS Synthetic Biology Page 30 of 31

1
2
3
4 490 (C)
5
6
7
8
9
10
11
12
13
14
15
16
17
18 491
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 30

ACS Paragon Plus Environment

Page 31 of 31 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 80x39mm (300 x 300 DPI)
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 Paragon Plus Environment