Cas9-Based Efficient Genome Editing in - ACS Publications

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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

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ACS Synthetic Biology

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CRISPR/Cas9-based efficient genome editing in Clostridium ljungdahlii, an

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autotrophic gas-fermenting bacterium

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He Huang†1, Changsheng Chai†1, Ning Li1, Pete Rowe2, Nigel P. Minton2, Sheng

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Yang1, 3, Weihong Jiang1, 3, and Yang Gu*1, 4

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Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai

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200032, China

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(SBRC), School of Life Sciences, University of Nottingham, Nottingham, NG7 2RD,

Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology,

Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre

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UK.

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3

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North Zhongshan Road, Nanjing 210009, China

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4

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Meilong Road, Shanghai 200237, China.

Jiangsu National Synergetic Innovation Center for Advanced Materials, SICAM, 200

Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130

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Corresponding author *Phone: 86-21-54924178. Fax: 86-21-54924015. E-mail: [email protected].

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Author Contributions

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†

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experiments. Y.G., H.H and N.P.M. designed the experiments and wrote the

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manuscript. P.R., S.Y. and W.J. assisted in experimental design and data analysis.

H.H. and C.C. contributed equally to this work. H.H, C.C and N.L. performed the

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ACS Paragon Plus Environment


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ABSTRACT:

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Acetogenic bacteria have the potential to convert single carbon gases (CO and CO2)

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into a range of bulk chemicals and fuels. Realization of their full potential is being

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impeded by the absence of effective genetic tools for high throughput genome

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modification. Here we report the development of a highly efficient CRISPR/Cas9

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system for rapid genome editing of Clostridium ljungdahlii, a paradigm for the

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commercial production of ethanol from synthesis gas. Following the experimental

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selection of two promoters (Pthl and ParaE) for expression of cas9 and the requisite

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single guide RNA (sgRNA), the efficiency of system was tested by making precise

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deletions of four genes, pta, adhE1, ctf and pyrE. Deletion efficiencies were

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100%, >75%, 100% and >50%, respectively. The system overcomes the deficiencies

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of currently available tools (more rapid, no added antibiotic resistance gene, scar-less

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and minimal polar effects) and will find utility in other acetogens, including pathogen

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Clostridium difficile.

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KEYWORDS: CRISPR/Cas9, rapid genome editing, strong promoters, Clostridium

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ljungdahlii, autotrophic bacterium, gas fermentation

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The use of fossil fuels is no longer tenable. A finite resource, their use is damaging the

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environment through pollution and global warming. Alternative, environmentally

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friendly sources of chemicals and fuels are required. To date the focus has been on

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using lignocellulose as a feedstock for microbial fermentation. However, its

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recalcitrance to deconstruction is making the development of economic processes

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extremely challenging. One alternative is to directly capture carbon before

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incorporation into lignocellulosic biomass. Acetogenic bacteria, typified by

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Clostridium ljungdahlii, are able to capture carbon (CO or CO2) through the

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Wood-ljungdahlii pathway, allowing them to grow on a spectrum of waste gases from

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industry (e.g., steel manufacture and oil refining, coal and natural gas) to produce

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ethanol (1-3). They can also consume ‘synthesis gas’ (mixtures of CO, CO2 and H2)

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made from the gasification of renewable resources, such as biomass in the form of

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domestic or agricultural waste. Acetogenic gas fermentation can, therefore, produce

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ethanol in any geographic region without competing for food or land resources (1-4).

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The real potential of acetogens, however, resides in their capacity to produce

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chemicals and fuels other than ethanol (5). This will require the redesign and

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implementation of more efficient metabolic pathways, adapting them to high

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performing manufacturing processes. The first tentative steps towards such goals have

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been made in recent years through the development and implementation of classical

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tools for the modification of acetogens such as Clostridium ljungdahlii and the closely

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related Clostridium autoethanogenum (5-9). The tools used to date, however, have

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been limited to either the introduction of autonomous plasmids encoding selected

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enzymes involved in product formation (5), the generation of insertional mutants

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using ClosTron mutagenesis (6) or the insertion of antibiotic resistance genes by

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homologous recombination (8) and the single crossover integration of suicide

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plasmids (9). In other work, a butyrate production from C. acetobutylicum was

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integrated into the C. ljungdahlii genome via homologous recombination, and

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thereafter modified by excision of the catP antibiotic resistance marker used for

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selection of the integrated DNA. The latter was achieved through the action of an

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introduced heterologous cre recombinase on loxP target sites that flanked the catP

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gene (7). Despite this progress, the developed methods and tools are in the main

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cumbersome and time consuming, being reliant on a number of labor intensive

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screening steps to identified the desired cell lines. Moreover, their deployment results

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in mutants with properties that are less than ideal. Thus, those mutants generate by

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single crossover integration of plasmids can revert through plasmid excision as a

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consequence of recombination between the duplicated regions of homology that

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mediated plasmid integration (9). On the other hand, the presence of inserted DNA in

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those mutants made using the ClosTron (6) or via double crossover integration of

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mutant alleles carrying antibiotic resistance genes (8), generate mutants that are not

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immune from polar effects on downstream genes. Moreover, even in those instances

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where the selectable marker is removed using Cre-LoxP technology, the final strain

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inevitably carries a scar in the chromosome (7).

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CRISPRs (clustered regularly interspaced short palindromic repeats) function as a

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prokaryotic acquired immune system, conferring resistance to exogenous genetic

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elements such as plasmids and phages (10). In type I and III systems, the pre-crRNA

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transcript is cleaved within the repeats by CRISPR-associated ribonucleases, releasing

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multiple small crRNAs. In type II systems, crRNA-tracrRNA hybrids complex with

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Cas9 to mediate interference. The CRISPR nuclease Cas9 is targeted by a short guide

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RNA that recognizes the target DNA via Watson-Crick base pairing. The guide

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sequence within these CRISPR RNAs can be easily replaced by a sequence of interest

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to retarget the Cas9 nuclease to a gene of choice. In recent years, the CRISPR-Cas9

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system has been developed into a powerful tool for high-efficiency genome editing

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(10, 11), by using a Cas9 nuclease, CRISPR RNA (crRNA) and trans-activating

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crRNA (tracrRNA), which work together to bind and cut at any target DNA site

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(12-14), or a simplified system, in which crRNA and tracrRNA were reprogrammed to

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yield a chimeric single guide RNA (sgRNA) that was more easily designed and

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manipulated (12, 15, 16). Recently, this powerful genetic tool has been harnessed for

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genome editing in two industrially relevant clostridial species, Clostridium

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cellulolyticum and Clostridium beijerinckii (17, 18). The former is a cellulolytic

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bacterium that is capable of directly converting lignocellulosic biomass into chemical

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commodities (19, 20), while the latter is highly effective at fermenting

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biomass-derived hydrolysates (21-23). Here we sought to establish a similar system in

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C. ljungdahlii, a paradigm for clostridial acetogens, based on a CRISPR/Cas9-based

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editing system developed in our laboratory for use in E.coli (24).

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As shown in Figures 1A and B, we constructed a plasmid that contained a sgRNA

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expression cassette and the Streptococcus pyogenes cas9, together with the requisite C.

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ljungdahlii-derived DNA required to mediate repair of the CRISPR/Cas9 catalyzed

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double strand break. For effective operation of the envisaged system, it is crucial that

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the incorporated CRISPR/Cas9 elements are well expressed in C. ljungdahlii under

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the growth conditions to be used. Accordingly, a number of heterologous promoters

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from C. acetobutylicum were cloned upstream of a promoter-less lacZ reporter gene,

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introduced into C. ljungdahlii on an autonomous plasmid, and the relative production

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of β-galactosidase was assayed at 24, 48 and 72 h time points during heterotrophic

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growth on fructose as the carbon source (Figure 2). Promoters from C. ljungdahlii

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were deliberately avoided to rule out unintended homologous recombination between

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the final plasmid and the chromosome. The promoters tested were the ParaE promoter

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of the C. acetobutylicum gene CA_C1339, together with the promoters of several

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solventogenic genes that had previously been characterized, namely, Pthl (25, 26), Pptb

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(25, 26), and Padc (26). The data obtained demonstrated that Pthl and ParaE promoters

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exhibited much greater activity at all three time points than Pptb and Padc. The Pthl and

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ParaE promoters were, therefore, selected to mediate expression of Cas9 and sgRNA,

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respectively, in C. ljungdahlii.

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Having established the basic components of the C. ljungdahlii CRISPR/Cas9

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system, a plasmid (pMTLcas-pta, Figure 1A) capable of targeting the pta

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(CLJU_c12770) gene encoding phosphotransacetylase was assembled and transferred

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into C. ljungdahlii by electroporation. In parallel, two control plasmids, pMTLcasc

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(lacking the pta homology arms) and pMTLHa (carrying the pta homology arms, but

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lacking Cas9 and sgRNA), were also electroporated into C. ljungdahlii (Table S2).

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Transformed cells were plated on YTF (27) solidified media supplemented with 5

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µg/mL thiamphenicol and those colonies visible after 3 days of incubation subjected

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to PCR screening in order to detect the desired double-crossover genotype. The

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primers were designed such that a 3,313-bp PCR amplified fragment would be

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generated from the wild-type pta allele, whereas the mutant pta allele would generate

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a 2,300-bp DNA fragment. Of the 8 and 10 colonies obtained in two independent

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experiments (Table 1) evidence for the presence of the desired pta deletion was

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obtained in all cases. In contrast, no pta-deletion events were observed in the

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electro-transformants that carried the control plasmids, indicating that the generation

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of mutants was specific to the presence of a fully functional CRISPR/Cas9 system. In

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a significant number of cases, however, the PCR screening suggested that the colonies

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were composed of both mutant and wild-type populations, as evidenced by the

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presence of both the 2,300-bp and 3,313-bp PCR- amplified DNA fragments (Table 1

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and Figure 1C). These data may suggest that, at this stage in the development of the

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system and protocol, that complete cleavage of the target site by the Cas9/sgRNA

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complex is not occurring. More importantly, however, several colonies represented

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pure, clonal populations of the desired mutant, as evidenced by the generation of only

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a 2,300-bp PCR-amplified DNA fragment, and the complete absence of the 3,313-bp

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DNA fragment associated with the wild-type genotype (Figure 1C). The 2,300-bp

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PCR-amplified DNA fragment was also extracted and sequenced to further confirm

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the desired deletion (Fig. S1 in supplementary data). These data show the desired

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deletion mutant can be isolated in a single step, requiring no further restreaking and

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screening to identify pure clonal sub-populations of mutants.

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Phenotypic characterization of a pta mutant revealed that the production of acetic

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acid had been substantially reduced compared to the wild-type strain (Figure 3). This

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is consistent with the central role of phosphotransacetylase in the acetate acid-forming

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pathway (28). It was also notable that the growth rate of the mutants was significantly

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impaired in comparison to the wild-type (Figure 3). The pta-dependent acetic

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acid-forming pathway represents a major ATP donor in C. ljungdahlii during syngas

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fermentation (5). Impairment of growth is, therefore, most likely a consequence of the

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reduction in the cells ability to generate ATP.

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To further exemplify the system, three further C. ljungdahlii genes were targeted.

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These were, adhE1 (CLJU_c16510, encoding a bifunctional aldehyde/alcohol

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dehydrogenase), ctf (CLJU_c39430, which encodes acyl-CoA transferase) and pyrE

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(CLJU_c35680, which encodes orotate phosphoribosyltransferase). In every instance,

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evidence for the presence of mutant populations in some, or all, of the transformant

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colonies was obtained by PCR screening. In contrast to the result with pta, some of

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the colonies, although a minority, appeared to be composed of entirely wild-type cells.

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Nevertheless, the majority of the colonies were composed of either mutant or a

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mixture of mutant and wild-type populations. In the case of ctf, all (21 in total) of the

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colonies screened were composed of mutant and wild-type cells. In the case of pyrE,

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the majority (15 out of 22) comprised mutant and wild-type cells, with one pure

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mutant and 6 wild-type colonies. In contrast, in the case of adhE1, no mixed colonies

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were evident. Rather the transformant colonies comprised 14 deletion mutants and 4

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wild-type cell lines.

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To address the issue of mixed populations, colonies carrying both mutant and

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wild-type cells from the ctf and pyrE deletion experiments were resuspended in 20 µL

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liquid YTF medium (supplemented with 5 µg/mL thiamphenicol) and re-streaked onto

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YTF agar plates (supplemented with 5 µg/mL thiamphenicol). Following two days of

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incubation, 15 well isolated individual colonies from either the ctf or pyrE mixed

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mutant cell lines, respectively, were selected for PCR screening. In all cases, a single

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PCR-amplified DNA fragment was obtained consistent with the mutant allele (Table

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2). These data indicate that, at least in these instances, pure mutant populations can

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easily be isolated from primary colonies composed of mixed mutant and wild-type

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alleles by one-step subculturing. This may suggest that the prolonged incubation of

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cells in the continued presence of the active, targeted Cas9/sgRNA plasmid leads to

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total conversion of cells to mutants. Alternatively, ctf and pyrE mutants may possess a

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growth advantage over the wild-type cells, leading to their predominance as well

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isolated colonies. All these three deletion events were further confirmed by

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sequencing (Fig. S1 in supplementary data)

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Phenotypic characterization of an adhE1 mutant revealed that the production of

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ethanol had been significantly reduced compared to the wild-type strain (Figure 4A).

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This is consistent with the essential role of bifunctional aldehyde/alcohol

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dehydrogenase in the ethanol-forming pathway (28). The ctf mutant M-ctf also

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showed impaired growth compared to the wild-type strain, leading to decreased

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production of acetic acid and ethanol (Figure 4B). The phenotypic effect of deleting

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ctf is in agreement with the previously reported result, in which ctf was inactivate by

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single-crossover homologous recombination (7). In addition, the deletion of pyrE

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resulted in the desired FOA-resistant cells, which can grow on solidified medium

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supplemented with FOA (Figure 4C).

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In summary, here we report the first demonstration of CRISPR/Cas9 genome

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editing in an industrially important clostridial acetogen, C. ljungdahlii. This genetic

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tool facilitates rapid and precise chromosomal manipulation in C. ljungdahlii and

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overcomes intrinsic limitations in those methods described to date for constructing

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mutant strains for industrial applications. Mutants made are scar-less and devoid of

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undesirable antibiotic resistance markers, an essential feature of chassis destined for

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process scale-up. The system should prove applicable to other autotrophic Clostridium

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species for efficient genome editing, both industrially important (e.g., C.

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autoethanogenum) and pathogens (e.g., Clostridium difficile). More importantly, our

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systems will form the basis of more elaborate implementations of Synthetic Biology

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approaches in acetogens, such as gene regulation, elucidating gene function, pathway

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engineering and more sophisticated cell factory designs.

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METHODS

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Strains, Media, and Reagents

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The E.coli host strain DH5α E. coli strain was used for plasmid cloning and

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maintenance. It was grown in LB medium supplemented with chloramphenicol (12.5

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µg/mL) when needed. The C. ljungdahlii strain used was DSM 13528. It was grown

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in YTF medium (27) or a modified ATCC medium 1754 (2-fold increased

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concentration of all the mineral elements; fructose was removed) with a headspace of

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CO-CO2-H2-N2 (56%/20%/9%/15%; pressurized to 0.2 MPa). In addition, 5 µg/mL of

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thiamphenicol (Wako Pure Chemical Industries, Osaka, Japan) was supplemented as

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needed for plasmid selection. KOD plus Neo and KOD FX DNA polymerase (Toyobo,

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Osaka, Japan) was used for high fidelity DNA amplification and PCR screening of the

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desired genotypes of C. ljungdahlii. The oligonucleotide primers used were

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synthesized by GenScript (Nanjing, China). Plasmids were constructed using

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conventional restriction digestion enzymes and ligase purchased from Thermo Fisher

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Scientific (USA）and Takara (Dalian, China), respectively, or assembled through the

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ClonExpress MultiS One Step Cloning Kit (Vazyme Biotech Co., Ltd, Nanjing,

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China). Plasmid isolation and DNA purification were performed with kits purchased

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from Axygen (Hangzhou, China).

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Plasmid Construction

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The CRISPR/Cas9 editing plasmid pMTLcas-pta was assembled from the

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following fragments: linear plasmid pMTL83151 (29) obtained by double digestion

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with SacI and XhoI; the native cas9 of Streptococcus pyogenes was obtained by PCR

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using the primers cas9fw/cas9rw and pCas as the template; the Pthl and ParoE

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promoters were obtained from Clostridium acetobutylicum using the primers

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pthlrw/pthlfw and paraEfw/paraErw, respectively; sgRNA (which targets the 20-nt

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target spacer of the pta gene) was obtained by PCR using the primers

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ptagRNAs/gRNAaster and pTargetF as template; the two homologous arms (HAs)

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that flank the open reading frame of pta were amplified from C. acetobutylicum

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genomic DNA using the primers ptauas/ptauas and ptads/ptadas.

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Here, every 20-nt target sequence (5'-N20NGG-3', where N represents any

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nucleotide) chosen for sgRNA targeting was examined in advance by alignment

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search (NCBI BLAST), aiming to ensure no sequence with high nucleotide acid

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sequence similarity in the genome and thus avoid the potential “off-target” in gene

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deletion. Then, the sgRNA scaffold and ParaE promoter were assembled by an overlap

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extension PCR, which resulted in the ParaE-sgRNA fragment. This fragment was then

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assembled with the above HAs fragments by a second round of overlap extension

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PCR, which yielded the fragment ParaE-sgRNA-HA. Thereafter, the linear

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pMTL83151

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ParaE-sgRNA-HA were assembled to give the CRISPR/Cas9 editing plasmid

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pMTLcas-pta using the ClonExpress MultiS One Step Cloning Kit (Fig. 1A). When

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preparing the other three plasmids for the adhE1, ctf and pyrE deletion, the

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pMTLcas-pta plasmid was used as the template, and the sgRNA and homologous

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arms cassette were changed accordingly.

vector,

and

DNA

fragments

encompassing

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Pthl,

cas9,

and

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To construct the pMTLcasc control plasmid (cas9+sgRNA, without HAs), the

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pMTLcas-pta plasmid was digested with XbaI and XhoI to remove the original

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sgRNA and HAs. The resulting linear vector was then assembled (using the

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ClonExpress MultiS One Step Cloning Kit) with a single pta-specific sgRNA

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fragment that was PCR-amplified from the pTargetF template using the primers

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ptagRNAs/gRNAasre. Similarly, the other control plasmid pMTLHa (HAs, without

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cas9 and sgRNA) was obtained by removing cas9 and sgRNA from pMTLcas-pta by

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NotI-SalI double digestion, followed by blunt-ending and self-ligation.

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The reporter plasmids were constructed as follows: the chloramphenicol

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acetyltransferase gene (catP) was amplified from the pMTL83151 plasmid with the

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catP-SacI-For/catP-ClaI-Rev primers and digested with SacI/ClaI. The cleaved

266

fragment was then ligated with pXY1 plasmid digested with the same restriction

267

enzymes, to yield the plasmid pLXY1. Next, the lacZ reporter gene was excised from

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pIMP1-Pthl-LacZ by BamHI-SmaI digestion and ligated with the pLXY1 plasmid

269

digested with the same restriction enzymes, to give the plasmid pLXY1-Pthl-LacZ.

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Subsequently, the Pptb, Padc, ParaE promoters were PCR-amplified using genomic DNA

271

of C. acetobutylicum ATCC 824 as template and Pptb-PstI-For/Pptb-BamHI-Rev,

272

Padc-PstI-For/Padc-BamHI-Rev, and ParaE-PstI-For/ParaE-BamHI-Rev as primers.

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This reaction was digested with PstI/BamHI and ligated into the pLXY1-Pthl-LacZ

274

plasmid, which was also digested with the same restriction enzymes to replace the

275

original Pthl promoter. The outcomes of this procedure were three new reporter

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plasmids, namely pLXY1-Pptb-LacZ, pLXY1-Padc-LacZ and pLXY1-ParaE-LacZ.

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Gene Deletion by CRISPR/Cas9 Plasmids

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C. ljungdahlii was grown anaerobically at 37°C. The gene deletion was achieved by

280

delivering the CRISPR/Cas9 editing plasmid into C. ljungdahlii by electroporation.

281

Electrocompetent cells were prepared according to the previously described protocol

282

(8). The electroporation procedure was slightly modified. Briefly, 200 µL of

283

electrocompetent cells were thawed on ice and then mixed with 4 µg of plasmid DNA

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recovered from E. coli DH5α for approximately 10 min. The mixture of

285

electrocompetent cells and plasmid DNA was then transferred into a 2 mm-gap

286

electroporation cuvette (Harvard Apparatus, MA, USA) and pulsed with parameters

287

set at 1.0 kV, 200 Ω and 50 µF using Gene Pulser Xcell microbial electroporation

288

system (Bio-Rad). The cells were recovered in 2 mL YTF medium supplemented with

289

0.4% L-cysteine for 6 h. Then, they were plated onto YTF agar plates (supplemented

290

with 5 µg/mL of thiamphenicol) at 37°C for approximately three days. When the

291

colonies were visible on the agar plate, the primers listed in the Supplementary Table

292

S2 were used for the PCR screening of the desired deletion events. Then, the

293

PCR-amplified DNA fragments were extracted and sequenced to further confirm the

294

deletions.

295 296

β-Galactosidase Assays

297

The plasmids pLXY1-Pthl-LacZ, pLXY1-Pptb-LacZ, pLXY1-Padc-LacZ, and

298

pLXY1-ParaE-LacZ were transferred into C. ljungdahlii by electro-transformation. C.

14


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299

ljungdahlii cells that harbored the reporter plasmids were grown anaerobically at

300

37°C and harvested at 24, 48 and 72 h by centrifugation at 12, 000 g for 5 min. The

301

cell pellets were dissolved in the B-PER reagent (Thermo Scientific Pierce®, USA)

302

and vortexed for 1 min for lysing the cells. The resulting cell lysate was heat-treated

303

at 60°C for 30 min to remove heat-unstable proteins and, then, centrifuged at 12, 000

304

g for 30 min. The supernatant was used for the β-galactosidase assays as previously

305

reported (25).

306 307

Phenotypic Identification of Cells with pyrE Deletion

308

Cells with pyrE deletion can be identified on growth medium supplemented with

309

5-fluoroorotic acid (FOA), as FOA is highly toxic to cells harboring pyrE-based

310

pyrimidine synthetic pathway. The assay was performed as previously described (30),

311

excepted that solidified YTF medium supplemented with 2 mg/mL FOA was used as

312

the assay medium.

313 314

Fermentation

315

All the C. ljungdahlii mutants were serially subcultured in YTF liquid medium (no

316

antibiotic added) to eliminate the plasmid before fermentation. The fermentation was

317

performed in 125 mL Wheaton serum bottles (Sigma-Aldrich, USA) with 30 mL

318

working volume. Briefly, 100 µL of frozen stock was inoculated into 5 mL YTF

319

liquid medium and incubated anaerobically at 37°C for 24 h. When the optical density

320

(OD600) of cells reached 0.5-1.0, approximately 5% (vol/vol) of the inoculum was

321

transferred into 30 mL of a modified ATCC medium 1754 (2-fold increased 15



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

322

concentration of all the mineral elements; fructose was removed) with a headspace of

323

CO-CO2-H2-N2 (56%/20%/9%/15%; pressurized to 0.2 MPa) for fermentation. The

324

bottles were incubated horizontally in a shaking incubator at 37°C and 150 rpm. The

325

culture was sampled every 24 h and, then, the gases were added into the headspace

326

(again at 0.2 MPa).

327

Analytical Methods

328

Cell growth was followed by measuring the absorbance of the sample at A600

329

(OD600) using a spectrophotometer (U-1800, Hitachi, Japan). The concentrations of

330

ethanol and acetate were measured by an internal standard method using gas

331

chromatography (7890 A, Agilent, Wilmington, DE, USA) equipped with a flame

332

ionization detector and a capillary column (Alltech ECTM-WAX). The analysis was

333

carried out under the following conditions: oven temperature, programmed from 85 to

334

150°C; injector temperature, 250°C; detector temperature, 300°C; nitrogen (carrier

335

gas) flow rate, 20 mL/min; hydrogen flow rate, 30 mL/min; air flow rate, 400 mL/min.

336

The internal standards were isobutanol, isobutyric acid and hydrochloric acid (for

337

acidification).

338

16


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339 340

ACKNOWLEDGEMENTS This work was funded by the National High-tech Research and Development

341

Program

of China

(2015AA020202),

the

Chinese

342

(KSZD-EW-Z-017-2 and KGZD-EW-606), the Synthetic Biology China-UK

343

Partnering Award (Utilizing Steel Mill 'Off-Gas' for Chemical Commodity Production

344

using Synthetic Biology), funded by the BBSRC (BB/L01081X/1) and the Chinese

345

Academy of Sciences, the National Natural Science Foundation of China (31370133

346

and 31121001) and Youth Innovation Promotion Association CAS.

17


Academy of

Sciences


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

347

SUPPORTING INFORMATION

348

Table S1 Strains and plasmids in this study

349

Table S2 Oligonucleotides used in this study

350

Fig. S1 Confirmation of the deletion of pta, adhE1, ctf and pyrE by

351

sequencing

18


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352

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carbon monoxide. Curr. Opin. Biotechnol. 22, 326-330.

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Wilkins, M. R., and Atiyeh, H. K. (2011) Microbial production of ethanol from

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ljungdahlii represents a microbial production platform based on syngas. Proc.

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Mock, J., Zheng, Y., Mueller, A. P., Ly, S., Tran, L., Segovia, S., Nagaraju, S.,

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Ueki, T., Nevin, K. P., Woodard, T. L., and Lovley, D. R. (2014) Converting

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mBio 5, e01636-01614.

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Leang, C., Ueki, T., Nevin, K. P., and Lovley, D. R. (2013) A genetic system

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Tremblay, P. L., Zhang, T., Dar, S. A., Leang, C., and Lovley, D. R. (2012) The

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CRISPR-Cas systems. Curr. Opin. Microbiol. 18, 83-89.

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Fineran, P. C., and Dy, R. L. (2014) Gene regulation by engineered

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Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and

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Charpentier, E. (2012) A programmable dual-RNA-guided DNA endonuclease

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in adaptive bacterial immunity. Science 337, 816-821.

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Jiang, W., Bikard, D., Cox, D., Zhang, F., and Marraffini, L. A. (2013)

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RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat.

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Deltcheva, E., Chylinski, K., Sharma, C. M., Gonzales, K., Chao, Y., Pirzada,

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

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Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J.

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Wang, Y., Zhang, Z. T., Seo, S. O., Choi, K., Lu, T., Jin, Y. S., and Blaschek, H.

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using CRISPR/Cas9 system. J. Biotechnol. 200, 1-5.

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

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Z., and Zhou, J. (2015) Efficient genome editing in Clostridium cellulolyticum

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via CRISPR-Cas9 nickase. Appl. Environ. Microbiol. 81, 4423-4431.

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Higashide, W., Li, Y., Yang, Y., and Liao, J. C. (2011) Metabolic engineering

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of Clostridium cellulolyticum for production of isobutanol from cellulose.

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Liao, J. C., Schadt, C. W., Guss, A. M., Yang, Y., and Graham, D. E. (2012)

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Combined inactivation of the Clostridium cellulolyticum lactate and malate

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dehydrogenase genes substantially increases ethanol yield from cellulose and

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Xiao, H., Li, Z., Jiang, Y., Yang, Y., Jiang, W., Gu, Y., and Yang, S. (2012)

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Metabolic engineering of D-xylose pathway in Clostridium beijerinckii to

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optimize solvent production from xylose mother liquid. Metab. Eng. 14,

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Wang, Y., and Blaschek, H. P. (2011) Optimization of butanol production from

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tropical maize stalk juice by fermentation with Clostridium beijerinckii

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NCIMB 8052. Bioresour. Technol. 102, 9985-9990.

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Du, T. F., He, A. Y., Wu, H., Chen, J. N., Kong, X. P., Liu, J. L., Jiang, M., and

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Ouyang, P. K. (2013) Butanol production from acid hydrolyzed corn fiber with

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Clostridium beijerinckii mutant. Bioresour. Technol. 135, 254-261.

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Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., and Yang, S. (2015) Multigene

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editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl.

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Environ. Microbiol. 81, 2506-2514.

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Girbal, L., Mortier-Barriere, I., Raynaud, F., Rouanet, C., Croux, C., and

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Soucaille, P. (2003) Development of a sensitive gene expression reporter

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acetobutylicum. Appl. Environ. Microbiol. 69, 4985-4988.

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Tummala, S. B., Welker, N. E., and Papoutsakis, E. T. (1999) Development

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acetobutylicum ATCC 824. Appl. Environ. Microbiol. 65, 3793-3799.

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Humphreys, C. M., McLean, S., Schatschneider, S., Millat, T., Henstra, A. M.,

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Annan, F. J., Breitkopf, R., Pander, B., Piatek, P., Rowe, P., Wichlacz, A. T.,

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Woods, C., Norman, R., Blom, J., Goesman, A., Hodgman, C., Barrett, D.,

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Thomas, N. R., Winzer, K., and Minton, N. P. (2015) Whole genome sequence

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and manual annotation of Clostridium autoethanogenum, an industrially

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relevant bacterium. BMC genomics 16, 1085.

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Nolling, J., Breton, G., Omelchenko, M. V., Makarova, K. S., Zeng, Q., Gibson,

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R., Lee, H. M., Dubois, J., Qiu, D., Hitti, J., Wolf, Y. I., Tatusov, R. L., Sabathe,

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F., Doucette-Stamm, L., Soucaille, P., Daly, M. J., Bennett, G. N., Koonin, E.

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V., and Smith, D. R. (2001) Genome sequence and comparative analysis of the

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solvent-producing bacterium Clostridium acetobutylicum. J. Bacteriol. 183,

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Heap, J. T., Pennington, O. J., Cartman, S. T., and Minton, N. P. (2009) A

452

modular system for Clostridium shuttle plasmids. J. Microbiol. Methods 78,

453

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454

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Heap, J. T., Ehsaan, M., Cooksley, C. M., Ng, Y. K., Cartman, S. T., Winzer,

455

K., and Minton, N. P. (2012) Integration of DNA into bacterial chromosomes

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23



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Page 24 of 31

Table 1. Results of the pta deletion in C. ljungdahlii

458

Plasmids

Elements

pMTLcasc pMTLHa pMTLcas-pta

Cas9+sgRNA Homologous arms Cas9+sgRNA+ Homologous arms

Experimental rounds

Results (M/P/W/T)*

Efficiency#

— — 1

N N 7/1/0/8

— — 100%

2

4/6/0/10

100%

459

*M: number of colonies that harbored mixed wild-type and gene-deleted cells; P:

460

number of colonies that harbored pure gene-deleted cells; W: number of colonies that

461

harbored pure wild-type cells; T: total number of colonies used for PCR screening.

462

#

Efficiency: probability of deletion events occurring, calculated as (M+P)/T×100%.

463

24


Page 25 of 31

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


464 465

Table 2. Results of the adhE1, ctf and pyrE deletion in C. ljungdahlii Gene targets

Deletion size (bp)

Experimental rounds

adhE1

2,600

ctf

1,200

1 2 1 2 1 2

pyrE

570

Results Before subculturing (M/P/W/T)*

Efficiency#

After subculturing (M/P/T)

Efficiency#

0/3/1/4 0/11/3/14 11/0/0/11 10/0/0/10 6/0/6/12 9/1/0/10

75% 78% 100% 100% 50% 100%

— — 0/5/5 0/10/10 0/7/7 0/8/8

— — 100% 100% 100% 100%

466

*M: number of colonies that harbored mixed wild-type and gene-deleted cells; P:

467

number of colonies that harbored pure gene-deleted cells; W: number of colonies that

468

harbored pure wild-type cells; T: total number of colonies used for PCR screening.

469

#

Efficiency: probability of deletion events occurring, calculated as (M+P)/T×100%.

470

25



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

471

Fig. 1. Design of the CRISPR/Cas9 system for deletion of the C. ljungdahlii pta gene.

472

(A) Strategy for the construction of the plasmid pMTLcas-pta. (B) Strategy for pta

473

deletion using pMTLcas-pta. The yellow star indicates the recognition site of the

474

Cas9-sgRNA complex in the coding region of pta. (C) PCR screening of the pta

475

deletion. The 3.3 and 2.3 kbp bands represent the wild-type and the pta deletion

476

genotype, respectively. M: marker; WT: wild-type strain.

477

26


Page 26 of 31

Page 27 of 31

478

Fig. 2. Comparison of the strength of the four heterologous promoters (Pthl, ParaE, Pptb

479

and Padc from C. acetobutylicum ATCC 824) in C. ljungdahlii. Pcontrol: no

480

promoter to drive lacZ expression. Data are representative of three replicates.

481

Pthl thl Pptb ptb Pcontrol control

1600 1400

ParaE araE Padc adc

1200 U/mg

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


1000 800 600 400 200 0 24

48 Time (h)

27


72


482

Fig. 3. Phenotypic effect after the pta deletion in C. ljungdahlii. WT: wild-type strain;

483

M-pta: pta-deleted mutant. Data are representative of triplicate cultures.

484

Growth

5.0

2.5

4.0

Concentration(g/L)

3.0

2.0 WT M-pta

1.5 1.0 0.5

Acetate

3.0

WT M-pta

2.0 1.0 0.0

0.0 0

24

48 Time (h)

72

96

0

24

Ethanol

1.2 Concentration (g/L)

Growth (OD 600)

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

Page 28 of 31

1.0 0.8 WT M-pta

0.6 0.4 0.2 0.0 0

24

48 72 Time (h)

28


96

48 72 Time (h)

96

Page 29 of 31

485

Fig. 4. Phenotypic effect after deletion of adhE1 (A), ctf (B) and pyrE (C) in C.

486

ljungdahlii. WT: wild-type strain; M-adhE1: adhE1-deleted mutant; M-pyrE:

487

pyrE-deleted mutant. Data from (A) and (B) are representative of triplicate cultures.

488

(A)

Concentration (g/L)

2.5 2.0 1.5 1.0 WT M-adhE1

0.5

48 72 Time (h)

4.0 3.0 2.0 1.0

WT M-adhE1 0

96

24

48 72 Time (h)

1.0 0.8 0.6 0.4 WT M-adhE1

0.2 0.0 0

96

24

48 72 Time (h)

96

(B) 3.0

5.0

Growth Concentration(g/L)

2.5 2.0 1.5 1.0

WT M-ctf

0.5

Acetate

4.0 3.0 2.0 WT M-ctf

1.0 0.0

0.0 0

24

48

0

72 96 120 144 168 192 Time (h)

1.2 Concentration (g/L)

Growth (OD 600)

489

24

1.2

0.0

0.0 0

Ethanol

Acetate

5.0

Concentration (g/L)

Growth 3.0 Growth (OD 600)

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


24 48

72 96 120 144 168 192 Time(h)

Ethanol

1.0 0.8 0.6 0.4

WT M-ctf

0.2 0.0 0

24

48

72 96 120 144 168 192 Time (h)

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 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

490

(C)

491

30


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