Rapid generation of universal synthetic promoters for controlled gene

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Letter

Rapid generation of universal synthetic promoters for controlled gene expression in both gas-fermenting and saccharolytic Clostridium species. Gaohua Yang, Dechen Jia, Lin Jin, Yuqian Jiang, Yong Wang, Weihong Jiang, and Yang Gu ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 13, 2017

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ACS Synthetic Biology

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Rapid generation of universal synthetic promoters for controlled gene expression

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in both gas-fermenting and saccharolytic Clostridium species

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Gaohua Yang1, Dechen Jia1, Lin Jin1, Yuqian Jiang2, Yong Wang1, Weihong Jiang1, 4,

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Yang Gu*1, 3

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1

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

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200032, China

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Davis, Sacramento, CA 95817, USA

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

Department of Biochemistry and Molecular Medicine, University of California at

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3

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Meilong Road, Shanghai 200237, China

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4

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North Zhongshan Road, Nanjing 210009, China

Shanghai Collaborative Innovation Center for Biomanufacturing Technology, 130

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

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ACS Paragon Plus Environment


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AUTHOR INFORMATION:

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Corresponding author

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

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300 Fenglin Road, Shanghai 200032, China.

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Author Contributions

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G. Y., D. J and L. J. performed the experiments. Y. G. and Y. J. wrote the manuscript. Y.

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G., W. J. and Y. W. designed the experiments and wrote the manuscript.

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Conflict of Interest

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The authors declare no competing financial interest.

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ABSTRACT:

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Engineering solventogenic clostridia, a group of important industrial microorganisms,

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to realize their full potential in biorefinery application is still hindered by the absence

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of plentiful biological parts. Here, we developed an effective approach for rapid

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generation of a synthetic promoter library in solventogenic clostridia based on a

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dual-reporter system (catP and lacZ) and a widely used strong promoter Pthl. The

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yielded artificial promoters, spanning two orders of magnitude, comprised two

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modular components (the core promoter region and the spacer between RBS and the

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translation-initiating code), and the strongest promoter had an over 10-fold-higher

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activity than the original Pthl. The test of these synthetic promoters in controlled

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expression of sadh and danK in saccharolytic C. acetobutylicum and gas-fermenting C.

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ljungdahlii, respectively, gave the expected phenotypes, and moreover, showed good

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correlation between promoter activities and phenotypic changes. The presented

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wide-strength-range promoters here will be useful for synthetic biology application in

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solventogenic clostridia.

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KEYWORDS: dual-reporter system, synthetic promoters, controlled gene expression,

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C. ljungdahlii, C. acetobutylicum

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Clostridium is a genus of Gram-positive bacteria, in which solventogenic clostridia

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are well known for their ability in using a wide range of cheap substrates, such as

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lignocellulosic materials as well as CO2/H2 or CO, to produce a variety of bulk

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chemicals1-3. To fully explore the potential of solventogenic clostridia in biorefinery

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application, engineering Clostridium strains for more efficient metabolic pathways are

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inevitably required. To this end, many molecular tools have been developed in

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clostridia recently, including the insertional mutagenesis by using ClosTron4,

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mutagenesis or the insertion of genes by homologous recombination5, and

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CRISPR-Cas9-based gene deletion and chromosomal gene integration6-9, enabling

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genetic manipulation of many Clostridium species. However, the biological parts

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available for synthetic biological and metabolic engineering of solventogenic

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clostridia to date are still very limited. For example, only very few constitutive

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promoters, especially strong promoters (e.g., thl and pta promoter)10-12, are available

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for expressing functional genes in solventogenic clostridia. This will inevitably

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impede efficient manipulations in synthetic biology and metabolic engineering of

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Clostridium species.

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Here, we sought to establish a platform for rapid generation of artificial promoters

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in solventogenic clostridia, especially strong promoters. It is known that the regions

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flanking the –35 and –10 sequences of bacterial promoters contribute much to

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promoter strength13, 14. Thus, tuning promoter activity can be realized by randomized

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these core regions, and by this way, the resulting promoter library would contain a

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plentiful of variations. Here, a dual-reporter system (catP and lacZ reporter gene,

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encoding chloramphenicol acetyltransferase and β-galactosidase, respectively) that is

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suitable for solventogenic clostridia was designed to enable a high-throughput

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screening and characterizing of promoters with various activities, thereby yielding a

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synthetic

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translation-initiating site (ATG) of the strongest artificial promoter in the library was

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further optimized, providing more synthetic expression parts with higher strength.

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Finally, the use of above synthetic parts for controlled gene expression was performed

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in saccharolytic C. acetobutylicum and gas-fermenting C. ljungdahlii, aiming to

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demonstrate their value in engineering solventogenic clostridia.

promoter

library.

Next,

the

spacer

between

RBS

and

the

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Here, Pthl, a widely used constitutive promoter in solventogenic clostridia11, 15, 16,

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was chosen as the candidate for constructing the synthetic promoter library. As shown

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in Figure 1A, we first designed a reporting system using two reporter genes, catP and

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lacZ, encoding chloramphenicol acetyltransferase and β-galactosidase, respectively.

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The reporter gene catP (yielding Cmr) here enables an initial high-throughput

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screening of promoters with various activities on agar plates that contained different

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concentrations of chloramphenicol; and the following lacZ-based β-galactosidase

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activity assay will give a more accurate measurement of the promoter activity (Figure

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1). Next, to determine the change scale of each nucleotide around –35 and –10 region

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of Pthl, the sequences of the core regions (–35 and –10 as well as the flanking region)

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of 18 already identified promoters (including Pthl) in Clostridium species (Table S1)

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were inputted into WEBLOGO (http://weblogo.berkeley.edu), yielding a 37-nt motif

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(Figure 2A), in which the “TTG” and “TATAAT” (representing the –35 and –10

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region, respectively) were highly conservative. Based on this motif, the following

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sequences within the promoter Pthl were randomized: the 5'-four nucleotides (TATA)

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adjacent to “TTG”, the 3'-three nucleotides (TAA) adjacent to “TATAAT”, as well as

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the intervening spacer between “TTG” and “TATAAT” (Figure 2B). For creating

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random mutagenesis in above three regions of Pthl, a pair of primers (thl-m/cat, as

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shown in Figure 1 and Table S3), in which the primer thl-m contains randomized

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spacer sequences, were used to initiate a megaprimer PCR (Figure 1). Then, a library

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of plasmids, in which every plasmid harbored a Pthl-derived artificial promoter plus a

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catP-lacZ cluster, was rapidly obtained, according to the procedure shown in Figure 1.

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To examine the randomness of the library, a certain number of plasmids were

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extracted and sequenced, and the results showed that all the promoters differed in

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their core regions (data not shown).

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Such a plasmid library was then introduced into C. acetobutylicum by

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electroporation method. After obtaining transformants, we picked out a certain

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number of colonies for a replica-plating on five agar plate supplemented with 100,

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200, 400, 800 and 1, 200 µg/mL chloramphenicol, respectively, for a preliminary

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partition of the promoters with different activities (Figure 1). After 48 h of incubation,

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CmR colonies were visible on each of the five independent agar plates; besides, the

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number of CmR colonies gradually decreased along with the increase in

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chloramphenicol concentration (data not shown), indicating a selection effect from

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antibiotic. Next, a total of 35 colonies were picked out from different agar plates for

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LacZ activity assay. As shown in Figure 3, these Pthl-derived promoters exhibited a

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wide strength distribution, ranging from 1/10 to 1.4-fold activity of the original

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promoter Pthl. The core region sequences of these 35 synthetic promoters were listed

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in Table S4. Moreover, the strength distribution of the promoters partitioned by the

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reporter gene catP (CmR) and lacZ gave a good correlation, thereby indicating a high

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reliability of this dual-reporter system in screening artificial promoters in

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solventogenic clostridia. However, it should be noted that only very few promoters

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exhibited higher activity than Pthl (Figure 3); even for the strongest promoter (1200-9)

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in this library, it only gave a 0.4-fold increased strength over Pthl.

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To address this problem, we used another strategy, namely optimizing the length

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between the RBS and the translational start code (LRTSC), which has been proven to

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be crucial for gene expression in Clostridium species17, 18. Thereby, the LRTSCs of the

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strongest promoter 1200-9 in the library was adjusted, yielding 11 new promoters

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with different LRTSCs (including an introduced BamHI-recognizing GGATCC site in

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the cloning process) (Figure 4A). Next, the reporter gene lacZ was placed downstream

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to assay the strength of these expression parts, showing that these 11 yielded synthetic

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promoters gave quite different strength. Out of these 11, nine showed higher activity

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than the promoter Pthl; especially 1200-9-9 and 1200-9-13, their activities were over

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10-fold higher than that of the original promoter Pthl (Figure 4B). Thus, a library that

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comprised abundant Pthl-derived synthetic promoters with large strength span was

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rapidly developed in solventogenic clostridia through a dual-reporter-based screening

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combined with optimizing LRTSCs of promoters.

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Since the promoter Pthl has been known to function in C. ljungdahlii6, an interesting

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question is whether these Pthl-derived synthetic promoters are also available for this

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gas-fermenting Clostridium species. To address this, we picked out four synthetic

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promoters in the library, namely 100-1, 200-1, 1200-4 and 1200-9-9, to compare their

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activities in C. ljungdahlii and C. acetobutylicum. Encouragingly, these four

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promoters showed a good correlation in activity between C. acetobutylicum and C.

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ljungdahlii (Figure 5A), thereby illustrating the potential broad applicability of this

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synthetic promoter library in Clostridium species.

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Finally, we attempted to evaluate the utility of these biological parts in expressing

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functional genes in C. acetobutylicum or C. ljungdahlii. For C. acetobutylicum, since

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it can natively produce acetone, the sadh gene, encoding a primary/secondary alcohol

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dehydrogenase that is responsible for catalyzing acetone to isopropanol in Clostridium

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beijerinckii19, 20, was introduced into C. acetobutylicum, aiming to yield isopropanol.

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The sadh gene was expressed under the control of the above mentioned four synthetic

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promoters (100-1, 200-1, 1200-4 and 1200-9-9) as well as the original promoter Pthl,

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ranging from weak to strong promoter strength (Figure 5A). As expected, the

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expression of sadh under the control of 100-1, 200-1, Pthl, 1200-4 and 1200-9-9 led to

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83.3, 90.7, 222.0, 277.0, and 4235.4 mg/L of isopropanol, respectively (Figure 5B),

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showing a certain correlation between the expression part activity and the isopropanol

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levels. Especially for the strongest 1200-9-9, it conferred 4235.4 mg/L of isopropanol

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in batch fermentation without pH control, which was much higher than the reported

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data (3.1 g/L) by using adc promoter19. For C. ljungdahlii, the dnaK gene

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(CLJU_c07980), encoding a potential heat shock protein (Hsp), was chosen for

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overexpression. The DnaK protein has been reported to be able to protect cells from

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negative

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microorganisms21-23. Thus, dnaK overexpression in C. ljungdahlii here was expected

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to increase strain robustness and then enable higher product formation. Also, above

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five synthetic promoters were used to drive the expression of dnaK, and a plasmid that

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contained no dnaK gene was set as the control. Encouragingly, all five

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dnaK-overexpressing strains exhibited enhanced ability in forming products (acetic

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acid and ethanol, two major products in C. ljungdahlii) from C1 gases (Figure 5C).

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Besides, a certain correlation can be observed between the product titer (acetic acid

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plus ethanol) and the strength of the promoters used in expressing dnaK (Figure 5C).

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The use of the strongest expression part 1200-9-9 in the library conferred the highest

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product titer (6.1 g/L, including 3.5 g/L acetic acid and 2.6 g/L ethanol), nearly 70%

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increase over that (2.5 g/L acetic acid plus 1.1 g/L ethanol) of the control (Figure 5C).

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Moreover, a slight change in the proportion of acetate versus ethanol can be observed

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after dnaK overexpression using some promoters (Figure 5C), which was especially

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evident for the strongest promoter 1200-9-9. This phenotypic change may be

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attributed to the importance of DnaK in stress resistance24, 25, enabling an increased

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ability of C. ljungdahlii in acetic acid tolerance and the following switch from acetic

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acid to ethanol. Altogether, the above results suggested the usefulness of these

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expression parts in engineering C. ljungdahlii.

effects

of

physiological

and

extracellular

stresses

in

many

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In summary, here we report an exemplar of rapid generating a Pthl-derived synthetic

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promoter library for solventogenic clostridia based on a dual-reporter system. In the

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library, many expression parts that comprised artificial promoters and the optimized

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spacers between the RBS and the translational start code exhibited significantly

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enhanced activities over the native strong promoter Pthl, and moreover, showed broad

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usefulness in expressing functional genes in both gas-fermenting C. ljungdahlii and

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saccharolytic C. acetobutylicum. This method as well as the manipulation scheme

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provided in this study should prove applicable to other Clostridium species for

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high-efficient synthesis and selection of wide-strength-range promoters, especially

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strong promoters. These elements will form the basis of more elaborate molecular

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manipulations of synthetic biology applications in clostridia, such as pathway

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construction and fine-tuning, gene function dissection and sophisticated network

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designs.

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Methods

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Strains, Media and Reagents

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All the E. coli and Clostridium strains were listed in Table S2. The E. coli strain

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DH5α was adopted for plasmid cloning and maintenance and was grown in LB

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medium supplemented with antibiotics when needed. The C. acetobutylicum and C.

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ljungdahlii strain used were ATCC 824 and DSM 13528, respectively. C.

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acetobutylicum ATCC 824 was grown in CGM medium for inoculum preparation or

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P2 medium for fermentation, while C. ljungdahlii DSM 13528 was grown in YTF

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medium or a modified ATCC medium 17546 with a headspace of CO−CO2−H2−N2

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(56%/20%/9%/15%; pressurized to 0.2 MPa). During cultivation of C. acetobutylicum

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and C. ljungdahlii strain, 10 µg/mL of erythromycin and 5 µg/mL of thiamphenicol

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(Wako Pure Chemical Industries, Osaka, Japan) were supplemented, respectively, as

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needed for plasmid selection.

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Library Construction

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Library construction was preformed as shown in Figure 1. Briefly, plasmid

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pIMP1-thl-catP-lacZ was used as the template for PCR amplification to obtain

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megaprimers that comprised randomized core region sequence of the thl promoter and

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the downstream catP gene, by using primers thl-m and cat (Table S3). Then, the

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above randomized megaprimers were used again for PCR using the plasmid

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pIMP1-thl-catP-lacZ as the template again, yielding a series of linear plasmids

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containing randomized spacer sequences and the dual-reporter gene cluster (catP and

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lacZ gene). Next, the linear plasmid DNA (25 µl) was digested with DpnI (2µl)

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overnight, and then recycled through dialysis using membrane filter (0.025 µm,

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VSWP). Finally, the recycled linear plasmid DNA (5 µl) was transformed into E. coli

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strain DH5α for plasmid gap repair, yielding a promoter library.

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To examine the sequence randomness of these synthetic promoters, the E. coli

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transformants were plated to isolate individual clones. Then, a certain number of E.

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coli isolates were picked out and grown in liquid LB medium supplemented with 100

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µg/L ampicillin. Cells were harvested by centrifugation for plasmid extraction. The

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extracted plasmid was used as the template for PCR amplification by using primers

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pIMP1-pr-s and cat. The resulting DNA fragments were confirmed by sequencing.

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Plasmid Construction

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All the plasmids used in this study were derived from pIMP1 and pXY1 (Table S2),

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which contains the pIM13 and pCB102 replicon, respectively. It was known that the

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pIM13 gives a plasmid copy number of ca. 8 in C. acetobutylicum26, whereas no

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published data on the pCB102. The construction of the plasmids containing

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Pthl-derived promoter, truncated SRTSCs and the reporter gene lacZ was as follows:

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the lacZ gene that was PCR-amplified from the plasmid placZFT by using the primers

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lacZ(BamH1)-Ps/lacZ(Sma1)-Pr was digested with SmaI/BamHI and then inserted

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into the plasmid pIMP1 and pXY1, which were digested with the same restriction

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enzymes, yielding the plasmid pIMP1-LacZ and pXY1-LacZ, respectively. Then, the

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promoter Pthl, P100-1, P200-1, P1200-4 were PCR-amplified from the plasmid

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pIMP1-Pthl-cat-LacZ,

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pIMP1-P1200-4-cat-LacZ, respectively, by using the primers thl (Pst1)-Ps/thl

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(BamH1)-Pr; The promoter P1200-9-9 were PCR-amplified from the plasmid

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pIMP1-P1200-9-cat-LacZ by using the primers thl (Pst1)-Ps/thl-9 (BamH1)-Pr. These

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five promoters (Pthl, P100-1, P200-1, P1200-4 and P1200-9-9) were digested with PstI/BamHI

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and then inserted into both the plasmid pIMP1-LacZ and pXY1-LacZ, which were

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digested with the same restriction enzymes, yielding the plasmid pIMP1-Pthl-LacZ,

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pIMP1-P100-1-LacZ,

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pIMP1-P1200-9-9-LacZ,

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pXY1-P1200-4-LacZ and pXY1-P1200-9-9-LacZ.

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The construction of the plasmid for expressing sadhE was as follows: sadhE was

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PCR-amplified from the plasmid pSADH (Table S2) by using the primers

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sadhE(BamHI)-P/sadhE (SmaI)-Pr. The yielding DNA fragment was digested with

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SmaI/BamHI

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pIMP1-P200-1-LacZ, pIMP1-P1200-4-LacZ and pIMP1-P1200-9-9-LacZ (Table S2), which

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were digested with the same restriction enzymes, generating the recombinant plasmid

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pIMP1-Pthl-SadhE, pIMP1-P100-1-SadhE, pIMP1-P200-1-SadhE, pIMP1-P1200-4-SadhE

247

and pIMP1-P1200-9-9-SadhE.

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The construction of the plasmid for expressing dnaK was as follows: dnaK

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(CLJU_c07980) was PCR-amplified from Clostridium ljungdahlii genome by using

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the primers DnaK (BamHI)-PS/DnaK (SmaI)-Pr. The yielding DNA fragment was

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digested with SmaI/BamHI and then inserted into pXY1-Pthl-LacZ, pXY1-P100-1-LacZ,

and

pIMP1-P100-1-cat-LacZ,

pIMP1-P200-1-cat-LacZ

pIMP1-P200-1-LacZ, pXY1-Pthl-LacZ,

then

inserted

pXY1-P100-1-LacZ,

into

pIMP1-Pthl-LacZ,

13


and

pIMP1-P1200-4-LacZ, pXY1-P200-1-LacZ,

pIMP1-P100-1-LacZ,


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pXY1-P200-1-LacZ, pXY1-P1200-4-LacZ and pXY1-P1200-9-9-LacZ (Table S2), which

253

were digested with the same restriction enzymes, generating the recombinant plasmid

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pXY1-Pthl-DnaK, pXY1-P100-1-DnaK, pXY1-P200-1-DnaK, pXY1-P1200-4-DnaK and

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pXY1-P1200-9-9-DnaK.

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Consensus Sequence Identification of promoters in C. acetobutylicum

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The Weblogo tool (http://weblogo.berkeley.edu/) was used to generate the consensus

259

sequence of the promoters in C. acetobutylicum. The sequences of 18 identified

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promoters in C. acetobutylicum (Table S1) were collected and therein the sequence

261

from the fourth nucleotide at 5' of the –35 region to the third nucleotide at 3' of the

262

–10 region were extracted and inputted into the Weblogo (the length of inputted

263

sequences were set as 37-nt; and “-” was added into the spacer between –35 and –10

264

region if the length of inputted sequences was less than 37-nt). Then, a consensus

265

sequence of these 18 promoters was generated.

266 267

Promoter Strength Distinguishing

268

For selecting promoters with different activities, the plasmids carrying mutant thl

269

promoters were extracted from the above E. coli library and then transformed into C.

270

acetobutylicum ATCC 824 by electroporation. The resulting C. acetobutylicum

271

transformants were challenged on agar plates (CGM medium) with 100, 200, 400, 800

272

and 1200 µg/mL chloramphenicol to isolate individual clones, which were then picked

273

for β-galactosidase assays.

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β-Galactosidase Assays

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The plasmids carrying lacZ gene were transferred into C. acetobutylicum or C.

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ljungdahlii by electro-transformation. C. acetobutylicum or C. ljungdahlii cells were

278

grown anaerobically at 37°C. When the OD600 of the culture reached 2.0, cells were

279

harvested by centrifugation at 12, 000 g for 5 min. The cell pellets were dispersed in

280

the B-PER reagent (Thermo Scientific Pierce, USA) and vortexed for 1 min. To

281

remove heat-unstable proteins, the resulting cell lysate was heat-treated at 60 °C for

282

30 min and then centrifuged at 12, 000 g for 40 min. The supernatant was collected

283

for assaying β-galactosidase activity according to previously reported method27.

284 285

Fermentation

286

Batch fermentations of C. acetobutylicum ATCC 824 were performed anaerobically in

287

100-mL serum bottles with 20-mL working volume at 37°C using D-glucose as the

288

carbon source and the detailed manipulations were as same as described before 28.

289

Batch fermentations of C. ljungdahlii strain DSM 13528 were carried out

290

anaerobically in 125-mL serum bottles (Sigma-Aldrich, USA) with 30 mL of working

291

volume. The detailed manipulations were as same as described before6.

292 293

Analytical Methods

294

The measurement of cell growth was based on the absorbance of the culture at A600

295

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

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

296

acetate and ethanol were measured by using gas chromatography (7890 A, Agilent,

297

Wilmington, DE, USA) equipped with a flame ionization detector and a capillary

298

column (Alltech ECTMWAX). The analysis was performed under the following

299

conditions: oven temperature, programmed from 85 to 150°C at a rate of 50°C/min in

300

the first stage and maintained in 150°C for 2.5 min, then programmed from 150 to

301

200°C at a rate of 100°C/min in the second stage and maintained in 200°C for 1.5 min;

302

injector temperature, 250°C; detector temperature, 300°C; nitrogen (carrier gas) flow

303

rate, 25 mL/min; hydrogen flow rate, 30 mL/min; air flow rate, 400 mL/min. The

304

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

305

acidification).

306

The concentration of isopropanol was analyzed using high performance liquid

307

chromatography (Agilent 1260 infinity) equipped with HPX-87H ion exclusion

308

column (300mm×7.8mm) and a refractive index detector (Agilent). The analysis was

309

performed as the following conditions: column temperature, 60°C; 0.05 mM sulfuric

310

acid as mobile phase at a flow rate of 0.6 mL/min.

16


Page 16 of 29

Page 17 of 29

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311

ACKNOWLEDGEMENT

312

This work was funded by the National Natural Science Foundation of China

313

(31630003 and 31421061), National High-tech Research and Development Program

314

of China (2015AA020202), the Youth Innovation Promotion Association CAS and the

315

Synthetic Biology China-UK Partnering Award (Utilizing Steel Mill “Off-Gas” for

316

Chemical Commodity Production using Synthetic Biology), funded by the BBSRC

317

(BB/L01081X/1) and the Chinese Academy of Sciences. We thank Dr. Hailin Meng

318

for thoughtful discussions on this work.

17



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

319

SUPPORTING INFORMATION

320

Table S1 Core regions of 18 identified promoters in C. acetobutylicum

321

Table S2: Strains and plasmids in this study

322

Table S3: Oligonucleotides used in this study

323

Table S4: Sequence analysis of the core regions of Pthl-derived artificial promoters

324

with different activities

325

Table S5: The sequence of the original thl promoter (containing the synthetic spacer

326

between RBS and the initial code ATG) and its derivative promoters that were listed

327

in Figure 2A.

18


Page 18 of 29

Page 19 of 29

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328

REFERENCES:

329

1. Tracy, B. P., Jones, S. W., Fast, A. G., Indurthi, D. C., Papoutsakis, E. T. (2012)

330

Clostridia: the importance of their exceptional substrate and metabolite diversity

331

for biofuel and biorefinery applications. Curr. Opin. Biotechnol. 23 (3), 364-81.

332

2. Kopke, M., Held, C., Hujer, S., Liesegang, H., Wiezer, A., Wollherr, A.,

333

Ehrenreich, A., Liebl, W., Gottschalk, G., Dürre, P. (2010) Clostridium ljungdahlii

334

represents a microbial production platform based on syngas. Proc. Natl. Acad. Sci.

335

U. S. A. 107 (29), 13087-92.

336

3. Xue, C., Zhao, J., Chen, L., Yang, S. T., Bai, F. (2017) Recent advances and

337

state-of-the-art strategies in strain and process engineering for biobutanol

338

production by Clostridium acetobutylicum. Biotechnol. Adv. 35 (2), 310-322.

339 340

4. Kuehne, S. A., Heap, J. T., Cooksley, C. M., Cartman, S. T., Minton, N. P. (2011) ClosTron-mediated engineering of Clostridium. Methods Mol. Biol. 765, 389-407.

341

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

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

343

plasmids without a counter-selection marker. Nucleic Acids Res. 40 (8), e59.

344

6. Huang, H., Chai, C., Li, N., Rowe, P., Minton, N. P., Yang, S., Jiang, W., Gu, Y.

345

(2016) CRISPR/Cas9-based efficient genome editing in Clostridium ljungdahlii,

346

an autotrophic gas-fermenting bacterium. ACS Synth. Biol. 5 (12), 1355-1361.

347

7. Wang, Y., Zhang, Z. T., Seo, S. O., Lynn, P., Lu, T., Jin, Y. S., Blaschek, H. P.

348

(2016) Bacterial Genome Editing with CRISPR-Cas9: Deletion, Integration,

349

Single Nucleotide Modification, and Desirable "Clean" Mutant Selection in

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Clostridium beijerinckii as an Example. ACS Synth. Biol. 5 (7), 721-32. 8. Nagaraju, S., Davies, N. K., Walker, D. J., Kopke, M., Simpson, S. D. (2016)

352

Genome

editing

of

Clostridium

353

Biotechnol. Biofuels 9, 219.

autoethanogenum

using

CRISPR/Cas9.

354

9. Li, Q., Chen, J., Minton, N. P., Zhang, Y., Wen, Z., Liu, J., Yang, H., Zeng, Z., Ren,

355

X., Yang, J., Gu, Y., Jiang, W., Jiang, Y., Yang, S. (2016) CRISPR-based genome

356

editing and expression control systems in Clostridium acetobutylicum and

357

Clostridium beijerinckii. Biotechnol. J. 11 (7), 961-72.

358

10. Stim-herndon, K. P., Petersen, D. J., Bennett, G. N. (1995) Characterization of an

359

acetyl-CoA C-acetyltransferase (thiolase) gene from Clostridium acetobutylicum

360

ATCC 824, Gene 154 (1), 81-5.

361

11. Siemerink, M. A., Kuit, W., Lopez Contreras, A. M., Eggink, G., van der Oost, J.,

362

Kengen, S. W. (2011) D-2,3-butanediol production due to heterologous expression

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of an acetoin reductase in Clostridium acetobutylicum. Appl. Environ. Microbiol.

364

77 (8), 2582-8.

365

12. Boynton, Z. L., Bennett, G. N., Rudolph, F. B. (1996). Cloning, sequencing, and

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expression of genes encoding phosphotransacetylase and acetate kinase from

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Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 62 (8),

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2758-66.

369 370 371

13. Hammer, K., Mijakovic, I., Jensen, P. R. (2006) Synthetic promoter libraries--tuning of gene expression. Trends Biotechnol. 24 (2), 53-5. 14. Rytter, J. V., Helmark, S., Chen, J., Lezyk, M. J., Solem, C., Jensen, P. R.

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Synthetic promoter libraries for Corynebacterium glutamicum. Appl. Microbiol.

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Biotechnol. 98 (6), 2617-23.

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15. Dong, H., Tao, W., Zhang, Y., Li, Y. (2012) Development of an

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anhydrotetracycline-inducible gene expression system for solvent-producing

376

Clostridium acetobutylicum: A useful tool for strain engineering. Metab. Eng. 14

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(1), 59-67.

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16. Borden, J. R., Jones, S. W., Indurthi, D., Chen, Y., Papoutsakis, E. T. (2010) A

379

genomic-library based discovery of a novel, possibly synthetic, acid-tolerance

380

mechanism in Clostridium acetobutylicum involving non-coding RNAs and

381

ribosomal RNA processing. Metab. Eng. 12 (3), 268-81.

382

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

383

dioxide to butyrate with an engineered strain of Clostridium ljungdahlii. MBio 5

384

(5), e01636-14.

385

18. Yang, Y., Lang, N., Yang, G., Yang, S., Jiang, W., Gu, Y. (2016) Improving the

386

performance of solventogenic clostridia by reinforcing the biotin synthetic

387

pathway. Metab. Eng. 35, 121-8.

388

19. Lee, J., Jang, Y. S., Choi, S. J., Im, J. A., Song, H., Cho, J. H., Seung do, Y.,

389

Papoutsakis, E. T., Bennett, G. N., Lee, S. Y. (2012) Metabolic engineering of

390

Clostridium

391

fermentation. Appl. Environ. Microbiol. 78 (5), 1416-23.

392 393

acetobutylicum

ATCC

824

for

isopropanol-butanol-ethanol

20. Dai, Z., Dong, H., Zhu, Y., Zhang, Y., Li, Y., Ma, Y. (2012) Introducing a single secondary

alcohol

dehydrogenase

into

butanol-tolerant

21


Clostridium


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

394

acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation.

395

Biotechnol. Biofuels 5 (1), 44.

396

21. Abdullah Al, M., Sugimoto, S., Higashi, C., Matsumoto, S., Sonomoto, K. (2010)

397

Improvement of multiple-stress tolerance and lactic acid production in

398

Lactococcus lactis NZ9000 under conditions of thermal stress by heterologous

399

expression of Escherichia coli DnaK. Appl. Environ. Microbiol. 76 (13), 4277-85.

400

22. Seyer, K., Lessard, M., Piette, G., Lacroix, M., Saucier, L. (2003) Escherichia coli

401

heat shock protein DnaK: production and consequences in terms of monitoring

402

cooking. Appl. Environ. Microbiol. 69 (6), 3231-7.

403

23. Selby, K., Lindstrom, M., Somervuo, P., Heap, J. T., Minton, N. P., Korkeala, H.

404

(2011) Important role of class I heat shock genes hrcA and dnaK in the heat shock

405

response and the response to pH and NaCl stress of group I Clostridium botulinum

406

strain ATCC 3502. Appl. Environ. Microbiol. 77 (9), 2823-30.

407

24. Lemos, J. A., Luzardo, Y., Burne, R. A. (2007) Physiologic effects of forced

408

down-regulation of dnaK and groEL expression in Streptococcus mutans. J.

409

Bacteriol. 189 (5), 1582-8.

410

25. Abdullah Al, M., Sugimoto, S., Higashi, C., Matsumoto, S., Sonomoto, K. (2010)

411

Improvement of multiple-stress tolerance and lactic acid production in

412

Lactococcus lactis NZ9000 under conditions of thermal stress by heterologous

413

expression of Escherichia coli dnaK. Appl. Environ. Microbiol. 76 (13), 4277-85.

414

26. Lee, S. Y., Mermelstein, L. D., Papoutsakis, E. T. (1993) Determination of plasmid

415

copy number and stability in Clostridium acetobutylicum ATCC 824. FEMS

22


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416

Microbiol. Lett. 108 (3), 319-23.

417

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

418

characterization of a gene expression reporter system for Clostridium

419

acetobutylicum ATCC 824. Appl. Environ. Microbiol. 65 (9), 3793-9.

420

28. Xiao, H., Gu, Y., Ning, Y., Yang, Y., Mitchell, W. J., Jiang, W., Yang, S. (2011)

421

Confirmation and elimination of xylose metabolism bottlenecks in glucose

422

phosphoenolpyruvate-dependent phosphotransferase system-deficient Clostridium

423

acetobutylicum for simultaneous utilization of glucose, xylose, and arabinose.

424

Appl. Environ. Microbiol. 77 (22), 7886-95.

425

23



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Page 24 of 29

426

Figure 1 Rapid construction of an artificial promoter library based on a

427

dual-reporter (catP-lacZ) system. In step I, a RBS region (AGGAGG) was

428

introduced in the front of lacZ. In step III, the brown color in primer thl-m represents

429

a randomized sequence, aiming to yield megaprimers that contain mutated thl

430

promoter by using PCR .

431 CatP

lacZ

Primer thl-m TAGGAGGTTAGTTAGA

Pthl

catP Primer cat

Pthl

Pptb

Pthl

pIMP1-ptbcatP-lacZ Em r

pIMP1-thlcatP-lacZ Em r

Ampr

Ampr

Step III: yielding megaprimers with mutated thl promoter by PCR

Step I and II: cloning thl promoter and catP-lacZ dual reporter system into plasmid

Pthl

Step VI: plasmid gap repair in E. coli

catP

Step IV: megaprimer PCR

Step V: linear plasmids

100 µg/mL

200 µg/mL

400 µg/mL

800 µg/mL

1000 µg/mL

432

Step VII: circular plasmids containing mutated thl promoters

Step VIII: mutated thl promoter library of C. acetobutylicum

Step IX: initial promoter screening on agar plates

24


Step X: LacZ activity assay

Page 25 of 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


433

Figure 2 Visualization of the core region (–35 and –10 as well as the flanking

434

region) consensus of the 18 identified promoters and the characteristics of the thl

435

promoter in C. acetobutylicum. (A) The 37-nt consensus generated from the 18

436

already reported promoters (the detailed sequences were listed in Table S1) in

437

solventogenic clostridia. The sequences inputted into WEBLOGO covered 5'-four

438

nucleotides to “TTG” (–35 region), 3'-three nucleotides to “TATAAT” (–10 region), as

439

well as the intervening spacer between “TTG” and “TATAAT” of these promoters. For

440

the promoters harboring less than 18-nt intervening spacer between “TTG” and

441

“TATAAT”, the missing part was supplemented by “-” when inputting the sequence.

442

(B) The –35 and –10 regions of the thl promoter are underlined. The base “G” in

443

green color and with arrow represented the transcription start site. The bases

444

highlighted in red represented the randomized regions of the thl promoter. The

445

ribosome binding site (BRS) “AGGAGG” was also underlined.

446

(A)

447 448 449 450 451 452 453 454

(B)

thl promoter:

–35 –10 TTTTTAACAAAATATATTGATAAAAATAATAATAGTGGGTATAATTAAGTTGTT AGAGAAAACGTATAAATTAGGGATAAACTATGGAACTTATGAAATAGATTGA AATGGTTTATCTGTTACCCCGTATCAAAATTTAGGAGGTTAGTTAGA 25



455

Figure 3 LacZ activity assay of the Pthl-derived artificial promoters with different

456

strength distribution. The column in brown color represents the original thl

457

promoter and its strength was set as 1.0. 100, 200, 400, 800 and 1200 represent the

458

concentrations of chloramphenicol used in the initial screening of promoters with

459

different strength distribution on agar plates.

460

1.6 461 462 463 464 465 466

1.4

Relative strength to Pthl (LacZ activity, U/mg)

1.2 1.0 0.8 0.6 0.4 0.2

467

0.0 468 469

100-1 100-2 100-3 100-4 100-5 200-1 200-2 200-3 200-4 200-5 200-6 200-7 400-1 400-2 400-3 400-4 400-5 400-6 400-7 800-1 800-2 800-3 800-4 800-5 800-6 thl 800-7 1200-1 1200-2 1200-3 1200-4 1200-5 1200-6 1200-7 1200-8 1200-9

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

Promoters

470 471 472 473 474 475 476

26


Page 26 of 29

Page 27 of 29

477

Figure 4 Construction of multiple expression parts from the Pthl-derived

478

strongest artificial promoter 1200-9 by optimizing the length between the RBS

479

and the translational start code. (A) Expression parts derived from the promoter

480

1200-9 and the relevant RBS region. The 6-nt sequence “GGATCC” upstream of the

481

initial code “ATG” was a BamHI-recognizing site introduced in the cloning process.

482

The 1200-9-27 here represented the original sequence between RBS and initial code

483

“ATG” of the promoter 1200-9. (B) LacZ reporter analysis for the strength of the 11

484

derived expression parts. The column in grey color represented the strength of the

485

original thl promoter and was set as 1.0 (the control).

486

(A)

1200-9-27 1200-9-25 1200-9-23 1200-9-21 1200-9-19 1200-9-17 1200-9-15 1200-9-13 1200-9-11 1200-9-9 1200-9-7

487 488 489 490

AGGAGG TTAGTTAGAGTCGACTCTAGAGGATCC ATG AGGAGG TTAGTTAGAGTCGACTCTA1..GGATCC ATG AGGAGG TTAGTTAGAGTCGACTC11...GGATCC ATG AGGAGG TTAGTTAGAGTCGAC1111GGATCC ATG AGGAGG TTAGTTAGAGTCG11111..GGATCC ATG AGGAGG TTAGTTAGAGT1111111GGATCC ATG AGGAGG TTAGTTAGA11111111.GGATCC ATG AGGAGG TTAGTTA1..11111111.GGATCC ATG AGGAGG TTAGT1.1..11111111.GGATCC ATG AGGAGG TTA1.1.1..11111111.GGATCC ATG AGGAGG T1.1.1.1..11111111.GGATCC ATG

Promoter 1200-9

RBS

lacZ

491 492

(B)

494 495 496 497 498

Relative activity

493

13 12 11 10 9 8 7 6 5 4 3 2 1 0

12 0 12 0-9 00 -2 -9 1 12 -23 0 12 0-9 thl 0 12 0-9-17 00 -2 12 -9 5 00 -2 12 -9- 7 12 00 19 1200- 9-7 9 0 12 0-9-15 00 -1 12 -9- 1 00 13 -9 -9

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


27



499

Figure 5 Comparison of the thl promoter-derived expression parts in expressing

500

functional genes in both C. acetobutylicum (CAC) and C. ljungdahlii (CLJU). (A)

501

Comparison of the activities of some expression parts between CAC and CLJU by

502

using LacZ reporter assay. The strength of the initial thl promoter in C.

503

acetobutylicum was set as 1.0 (the control). The sampling time for LacZ reporter

504

assay of C. acetobutylicum and C. ljungdahlii was 24 and 48 h, respectively, ensuring

505

an optical density (OD600) of 2.0. (B) Isopropanol production by using multiple

506

expression parts to drive sadh gene in CAC. (C) Acetate and ethanol production by

507

overexpression of dnaK gene under the control of multiple expression parts in CLJU.

508 (A)

100

CAC

509 Relative activity

CLJU

510 511

10

1

512

0.1

513

0.01 1 010

514

9 900 12

7 Product concentration (g/L)

10

Acetate

6

Ethanol

5 4 3 2 1


1 20 0-

th l 12 00 -4 12 00 -9 -9

1

tro l Co n

00 -9 -9 12

04

28

10 0-

0

1

12 0

520

100

th l

519

1000

01

518

-4 00 2 1

(C)

20

517

l th

1 020

10000

01

516

(B)

10

515

Isopropanol concentration (mg/L)l

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 29

Page 29 of 29

Dual-reporter system

CatP

Pthl

Megaprimer PCR-based random mutagenesis of thl promoter

lacZ

TAGGAGGTTAGTTAGA

Expression of functional genes 7

10000

100 10

1.6 1.4

5 Relative strength

Product (g/L)

1000

Rapid screening for synthetic promoters

Acetate Ethanol

6

4 3 2 1

1

1.2 1.0 0.8 0.6 0.4

th l 00 12 -4 00 -9 -9 12

01

01 20

10

ro l

0.2

on t C

1

th 12 l 00 1 2 -4 00 -9 -9

1

20 0-

0

10 0-

Isopropanol (mg/L)

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


0.0 Promoters


Rapid generation of universal synthetic promoters for controlled gene

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