Enhancement of solvent production by overexpressing key genes of the acetone-butanol-ethanol fermentation pathway in Clostridium saccharoperbutylacetonicum N1-4

Accepted Manuscript Enhancement of solvent production by overexpressing key genes of the Acetone-Butanol-Ethanol fermentation pathway in Clostridium saccharoperbutylacetonicum N1-4 Shaohua Wang, Sheng Dong, Yi Wang PII: DOI: Reference:

S0960-8524(17)31572-9 http://dx.doi.org/10.1016/j.biortech.2017.09.024 BITE 18852

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

4 August 2017 3 September 2017 4 September 2017

Please cite this article as: Wang, S., Dong, S., Wang, Y., Enhancement of solvent production by overexpressing key genes of the Acetone-Butanol-Ethanol fermentation pathway in Clostridium saccharoperbutylacetonicum N1-4, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.09.024

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Enhancement of solvent production by overexpressing key genes of the Acetone-

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Butanol-Ethanol fermentation pathway in Clostridium saccharoperbutylacetonicum

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N1-4

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Shaohua Wanga, Sheng Donga, Yi Wanga,b,*

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a

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b

Department of Biosystems Engineering, Auburn University, Auburn, AL 36849, USA Center for Bioenergy and Bioproducts, Auburn University, Auburn, AL 36849, USA

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*

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Yi Wang,

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Department of Biosystems Engineering,

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Auburn University,

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215 Tom E. Corley Building,

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Auburn, AL, 36849 USA

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Tel: 1-334-844-3503

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Fax: 1-334-844-3530

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E-mail: [email protected]

Corresponding author:

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Abstract

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Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol

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producing strain. However, little information is available concerning its butanol production

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mechanism and the development of more robust strains. In this study, key biosynthetic

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genes (either endogenous or exogenous) including the sol operon (bld-ctfA-ctfB-adc),

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adhE1, adhE1D485G, thl, thlA1V5A, thlAV5A and the expression cassette EC (thl-hbd-crt-bcd)

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were overexpressed in C. saccharoperbutylacetonicum N1-4 to evaluate their potential in

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enhancement of butanol production. The overexpression of sol operon increased ethanol

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production by 400%. The overexpression of adhE1 and adhED485G resulted in a 5.6- and

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4.9-fold higher ethanol production, respectively, producing final acetone-butanol-ethanol

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(ABE) titers (30.6 and 30.1 g L-1) of among the highest as ever reported for solventogenic

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clostridia. The most significant increase of butanol production (by 13.7%) and selectivity

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(73.7%) was achieved by the overexpression of EC. These results provides a solid

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foundation and essential references for the further development of more robust strains.

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Keywords

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Clostridium saccharoperbutylacetonicum; metabolic engineering; butanol; sol operon;

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adhE; expression cassette (EC)

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1. Introduction Biobutanol produced through the clostridial acetone-butanol-ethanol (ABE)

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fermentation has been considered as one of the most promising biofuels and a valuable

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biochemical (Buehler & Mesbah, 2016; Jang et al., 2012b; Lee et al., 2008b). Clostridium

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strains employed for ABE production are mainly from the following four species: C.

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acetobutylicum, C. beijerinckii, C. saccharobutylicum, and C. saccharoperbutylacetonicum

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(Lee et al., 2008b). It has been well known that the ABE fermentation can be divided into

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two typical phases: acidogenesis and solventogenesis phases (Zhang et al., 2016). During

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the acidogenesis phase, acetic acid and butyric acid are produced as the main products

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along with the cell growth. Then during the solventogenesis phase, these acids are

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assimilated along with the consumption of additional carbon sources to produce acetone,

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butanol and ethanol. The key metabolic pathways and the related genes in the biphasic

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fermentation process have been well studied in C. acetobutylicum (Bennett & Rudolph,

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1995; Crown et al., 2011; Jones & Woods, 1986; Lee et al., 2008a). In the acidogenic

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phase, phosphotransacetylase (Pta) and acetate kinase (Ack) are responsible for the acetic

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acid production from acetyl-CoA. Four enzymes include thiolase (Thl), β-hydroxybutyryl-

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CoA dehydrogenase (Hbd), crotonase (Crt), and butyryl-CoA dehydrogenase (Bcd) are

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responsible for the conversion of acetyl-CoA to butyryl-CoA which will then be catalyzed

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by phosphotransbutyrylase (Ptb) and butyrate kinase (Buk) to generate butyric acid.

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During the solventogenesis, acetic and butyric acids are re-assimilated by acetoacetyl-

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CoA:acyl-CoA transferase (CtfA/B). Along with the re-assimilation of acids, acetoacetate

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is produced followed by being transformed to acetone through the catalysis by acetoacetate

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decarboxylase (Adc). The final end products ethanol and butanol are mainly produced

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under the action of the bifunctional protein AdhE (aldehyde/alcohol dehydrogenase). Two

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adhE genes, adhE1 and adhE2, both carried by the pSOL1 megaplasmid, as well as several

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bdh genes (bdhA, bdhB and bdhC, encoding butanol dehydrogenase) have been reported to

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play different roles in butanol production (Girbal & Soucaille, 1998; Yoo et al., 2016). In

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C. acetobutylicum, adhE1, ctfA and ctfB form the sol operon while adc is expressed alone

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in the opposite direction of the sol operon (Fischer et al., 1993; Nair et al., 1994). However,

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in C. beijerinckii NCIMB 8052, the sol operon is constituted by ald (aldehyde

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dehydrogenase encoding gene), ctfA, ctfB and adc (Chen & Blaschek, 1999).

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To improve the butanol production, metabolic flux in the alcohol biosynthesis pathways

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has been enhanced through metabolic engineering in solventogenic clostridial strains. The

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butanol titer was increased by 2.8 g L-1 when the buk gene was disrupted in C. beijerinckii

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(Wang et al., 2013). In C. acetobutylicum, the butanol production was improved by 160%

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to 18.9 g L-1 through overexpressing the adhE1D485G gene, encoding a mutated

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aldehyde/alcohol dehydrogenase, based on the mutant in which both the pta and buk genes

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were disrupted (Jang et al., 2012a). Recently, Lee et al. overexpressed the thl, adhE1, and

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ctfA/B genes in the C. acetobutylicum mutant strain whose pta and buk genes were

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knocked out, resulting in an extremely high butanol productivity (2.64 g L-1h-1) in a long-

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term fermentation (Lee et al., 2016). By overexpressing the expression cassette EC

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including the hbd, thl, crt and bcd genes as well as the adhE and ctfA/B genes in C.

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acetobutylicum, the butanol production was improved from 8.27 g L-1 to 14.86 g L-1 (Hou

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et al., 2013).

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C. saccharoperbutylacetonicum N1-4 (ATCC 13564) is well-known as a hyper-butanolproducing strain (Motoyoshi, 1960). Though the favorable fermentation characteristics

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have been broadly studied (Al-Shorgani et al., 2012; Tashiro et al., 2007; Thang et al.,

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2010), metabolic engineering of this strain is apparently lagging compared to C.

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acetobutylicum and C. beijerinckii. Recently, the genome sequence of C.

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saccharoperbutylacetonicum ATCC 27021 (a lysogenic derivative strain of ATCC 13564)

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has been published (del Cerro et al., 2013; Keis et al., 1995). Similar with C. beijerinckii

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NCIMB 8052, the sol operon of C. saccharoperbutylacetonicum includes four genes (bld,

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ctfA, ctfB and adc), and the transcription of them is reported to be essential for solvent

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production (Kosaka et al., 2007). The corresponding genes including hbd, thl, crt and bcd

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reported as the expression cassette EC (Hou et al., 2013) are also annotated on the genome

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(NC_020291). The genome of C. saccharoperbutylacetonicum has a size of 6.666 Mb (the

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largest genome for all the known solventogenic clostridial strains) and demonstrates a high

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multiplicity for many genes related to ABE fermentation. Until now, there is a lack of

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information concerning the function of the specific genes related to butanol production in

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C. saccharoperbutylacetonicum. Furthermore, little work has been done to develop more

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robust C. saccharoperbutylacetonicum strains for butanol production. Recently, a plasmid

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transformation protocol for C. saccharoperbutylacetonicum was reported by Herman et al.

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(Herman et al., 2016). Moreover, a CRISPR-Cas9 based efficient genome editing system

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has been recently developed in our group (Wang et al., 2017). The availability of the

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genome information and newly developed genetic engineering tools pave the way for the

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metabolic engineering of C. saccharoperbutylacetonicum.

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In this study, various key genes in the butanol biosynthesis pathway including the sol

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operon (bld-ctfA-ctfB-adc), adhE1, adhE1D485G, thl, thlA1V5A, thlAV5A and the expression

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cassette EC (thl-hbd-crt-bcd) were overexpressed in C. saccharoperbutylacetonicum N1-4.

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The recombinant strains were characterized through ABE fermentation to evaluate the

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effects of different gene overexpression strategies on butanol production. This study

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provides a solid foundation for clarifying roles of the key genes in the solvent biosynthesis

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pathway and further developing more robust C. saccharoperbutylacetonicum strains for

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solvent production through metabolic engineering strategies.

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2. Materials and methods

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2.1. Bacterial strains, plasmids, oligonucleotides, and culture conditions

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All the strains, plasmids and the selected genes used in this study are listed in Table 1;

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C. saccharoperbutylacetonicum N1-4 was grown in an anaerobic chamber (N2-CO2-H2

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with a volume ratio of 85:10:5) at 35 ºC in tryptone-glucose-yeast extract (TGY) medium

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(Wang et al., 2017; Wang et al., 2013). When appropriate, clarithromycin (Cla) was added

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into TGY medium to a final concentration of 30 µg mL-1. E. coli ER2523 (NEB

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Express) was used for DNA cloning. It was grown aerobically at 37 ºC in Luria-Bertani

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(LB) medium supplemented with 100 µg ml-1 of ampicillin (Amp) as needed.

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2.2. Construction of recombinant plasmids

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The plasmid pYW25 is derived from pTJ1 with the thiolase promoter (Pthl), two BseRI

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sites, and thiolase terminator (Tthl) inserted sequentially between the ApaI and BamHI

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restriction enzyme sites (Wang et al., 2013). Either pTJ1 or pYW25 was used as the

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mother vector for the recombinant plasmid construction. The Phanta Max Super-Fidelity

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DNA Polymerase (Vazyme Biotech Co., Ltd., Nanjing, China) was used for the PCR to

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amplify DNA fragments for cloning purposes. The sol operon including bld, ctfA, ctfB and

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adc (Kosaka et al., 2007) was amplified from C. saccharoperbutylacetonicum N1-4 using

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primers YW1075 and YW1076, followed by being inserted into the BseRI sites of pYW25,

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generating pSH1. The adhE1 gene from C. acetobutylicum ATCC 824 was amplified with

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primers YW1100 and YW1101. The ferredoxin promoter (Pfd) was employed for the

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overexpression of adhE1 and was amplified from C. saccharoperbutylacetonicum N1-4

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using primers YW1098 and YW1099. After being fused together through

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overlapping extension PCR (SOE-PCR) with primers YW1098 and YW1102, Pfd and

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adhE1 was inserted into the ApaI site of pYW25, generating pSH2. To obtain adhE1D485G

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(by introducing a single nucleotide mutation to adhE1) with the promoter (Pfd), two

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fragments were amplified first from pSH2 with primer pairs of YW1098 and YW1103, and

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of YW1104 and YW1102, respectively. Then the desirable Pfd-adhE1D485G was generated

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through SOE-PCR with primers YW1098 and YW1102, followed by being inserted into

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the ApaI site of pYW25, generating pSH3. The recombinant plasmid pSH4 was

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constructed through the insertion of thl along with its own promoter (amplified from C.

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saccharoperbutylacetonicum N1-4 using primers YW1238 and YW1239) into the EcoRI

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site of pTJ1. The mutant of thl, thlA1V5A derived from C. saccharoperbutylacetonicum N1-

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4 was amplified in two steps: first round of PCR using primer pairs of YW1238 &

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YW1904 and YW1239 & YW1905, and then SOE-PCR with primers of YW1238 &

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YW1905. Similarly, the mutant thlAV5A was amplified from C. acetobutylicum ATCC824

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using primer pairs of YW1906 & YW1907 and YW1908 &YW1909 for the first round of

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PCR, and then using YW1906 & YW1909 for the SOE-PCR. Subsequently, thlA1V5A and

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thlAV5A were inserted into the EcoRI site of pTJ1, generating pSH4 and pSH5, respectively.

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Based on the plasmid pSH4 with the original thiolase gene of C.

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saccharoperbutylacetonicum N1-4, other three genes from the expression cassette EC (hbd,

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crt and bcd) were added to construct pSH7. The hbd gene was amplified with primers

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YW1240 and YW1367. While crt and bcd genes were amplified as a single fragment using

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primers YW1368 and YW1369. Then, pSH7 was obtained through the successive insertion

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of these two fragments into the KpnI site and the BamHI site of pSH4 respectively.

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2.3. Transformation and recombinant strains verification

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The plasmids pSH1, pSH2, pSH3, pSH4, pSH5 and pSH6 as well as the control vector

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pTJ1 were transformed into C. saccharoperbutylacetonicum N1-4 through electroporation

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as described in our previous work (ice-cold condition) using a Gene Pulser Xcell

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electroporation system (Bio-Rad Laboratories, Hercules, CA) (Wang et al., 2017). While

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the initial transformation of pSH7 was unsuccessfully with the same protocol, a

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transformation procedure at room temperature as described by Herman et al. (Herman et al.,

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2016) was applied (with 1000 V of voltage, 25 µF of capacitance and 300 Ω of resistance)

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and pSH7 was ultimately transformed into C. saccharoperbutylacetonicum N1-4. After

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electroporation, following the incubation for about 10-12 h (‘ice-cold condition’ protocol)

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or 2-4 h (‘room temperature condition’ protocol), the recovered cells were spread onto

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TGY agar plates with Cla and incubated at 35 ºC under anaerobic conditions until colonies

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were observed (about 24 hours after the transformation). Single colonies were randomly

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picked and the presence of plasmid was verified using colony PCR with primers YW880

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and YW881 targeting on the erythromycin resistance gene contained in all the used

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plasmids. The generated recombinant strains were named according to their harbored

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plasmid as C. saccharoperbutylacetonicum pSH1, C. saccharoperbutylacetonicum pSH2,

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C. saccharoperbutylacetonicum pSH3, C. saccharoperbutylacetonicum pSH4, C.

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saccharoperbutylacetonicum pSH5, C. saccharoperbutylacetonicum pSH6, C.

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saccharoperbutylacetonicum pSH7 and C. saccharoperbutylacetonicum pTJ1, respectively.

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2.4. Fermentation Bach fermentation was performed in BioFlo 115 benchtop bioreactors (New Brunswick

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Scientific Co., Enfield, CT) with a working volume of 1.5 liters (Wang et al., 2017). P2

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medium (Wang et al., 2013) with 80 g l-1 glucose as the carbon source (supplemented with

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2 g l-1 yeast extract and 6 g l-1 tryptone) was used for the fermentation. The anaerobic

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condition was generated by sparging oxygen-free nitrogen through the fermentation broth

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starting several hours before the inoculation until the cell culture initiates its own gas

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production.

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The glycerol stock of the recombinant strain containing either the gene-overexpressing

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plasmid or the control vector (pTJ1) was inoculated into TGY medium containing 30 µg

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mL-1 Cla and cultivated anaerobically to prepare the seed culture. When the OD600 reached

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~0.8, the culture was inoculated into the reactor at an inoculum ratio of 5% (vol/vol).

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Meanwhile, Cla was added into the reactor to a final concentration of 30 µg mL-1.

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Fermentation was performed at 30 ºC with 50 rpm agitation. 1 M NaOH was used to

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control the pH above 5.0 throughout the fermentation process. All fermentations were

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performed in duplicate.

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2.5. Analytical methods

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Fermentation characteristics were analyzed as described in our previous report (Wang et

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al., 2017). An Ultrospec 10 cell density meter (Amersham Biosciences Corp., Piscataway,

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NJ) was used for cell density (OD600) measurement. Concentrations of glucose and

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fermentation products including ABE (acetone, butanol and ethanol) and acids (acetate,

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butyrate) were determined using a high-performance liquid chromatography (Agilent

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Technologies 1260 Infinity series, CA) with a refractive index Detector (RID), equipped

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with a Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA). H2SO4 (0.005 N)

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was used to elute the column with a flow rate of 0.6 ml min-1 at 25 ºC.

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3. Results and discussions

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3.1. Effects of the overexpression of sol operon on butanol production

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The kinetics of batch fermentation with C. saccharoperbutylacetonicum pSH1 and C.

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saccharoperbutylacetonicum pTJ1 are illustrated in Figure 1. C.

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saccharoperbutylacetonicum pSH1 consumed glucose slowly and left more glucose (23.7

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g L-1) than C. saccharoperbutylacetonicum pTJ1 (19.0 g L-1). However, as shown in Table

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2, a higher maximum OD600 (12.8) was observed with C. saccharoperbutylacetonicum

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pSH1 than the control strain (11.8). The overexpression of the sol operon in C.

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saccharoperbutylacetonicum pSH1 led to a complete re-assimilation of acetate and

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butyrate (Figs. 1B and 1C). Unlike that in C. saccharoperbutylacetonicum pTJ1 which

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demonstrated an acetate peak at about 18 h, the acetate in C. saccharoperbutylacetonicum

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pSH1 decreased continually, and reached and kept at zero level after 42 h. Similarly, the

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butyrate concentration in C. saccharoperbutylacetonicum pSH1 was maintained at zero

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level throughout the whole fermentation process. Besides the ctfA/B overexpressed in this

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study, there are another annotated ctfA/B gene cluster (ctfA2, CSPA_RS10270 and ctfB2,

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CSPA_RS10275) in the genome of C. saccharoperbutylacetonicum N1-4. However, it was

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reported that the overexpression of ctfA2/B2 led to the accumulation of acetate and

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butyrate while the deletion of them did not significantly affect the ABE fermentation

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(Herman et al., 2016). Taken together of the results from both studies, we can conclude

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that the ctfA/B within the sol operon rather than the ctfA2/B2 are the primary CoA-

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transferase genes that are responsible for the acid re-assimilation in C.

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saccharoperbutylacetonicum.

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Despite the efficient acid re-assimilation in C. saccharoperbutylacetonicum pSH1, the

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acetone production in this strain decreased by 1.0 g L-1 compared to the control strain (Fig.

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1D and Table 2). However, the ethanol production in C. saccharoperbutylacetonicum

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pSH1 reached 6.0 g L-1, representing a 400% improvement than the control. Similar results

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were demonstrated when a single adh11 (equal to bld) was overexpressed in the same

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strain (Herman et al., 2016). It was expected that the overexpression of bld would enhance

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the conversion of butyryl-CoA to butyraldehyde, thus leading to increased butanol

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production. However, surprisingly the overexpression of sol operon in C.

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saccharoperbutylacetonicum pSH1 led to a 17.6% decrease in butanol production (vs. the

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control). The tremendous increase of ethanol production in C. saccharoperbutylacetonicum

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pSH1 indicated that the carbon flow from acetyl-CoA is tending to be converted to ethanol

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rather than to be converted to butryl-CoA. In fact, the enhanced ethanol production through

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the overexpression of sol operon (particularly bld) might have impaired the carbon flow

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from acetyl-CoA to the longer chain metabolites (namely, acetoacetyl-CoA, and further to

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butyryl-CoA), and thus decreased the butanol and acetone production. Furthermore, the

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elevated ethanol production and decreased butanol production in C.

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saccharoperbutylacetonicum pSH1 than in the control suggested that bld might have a

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better affinity for acetylaldehyde (and further ethanol) production rather than for

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butyraldehyde (and further butanol) production. However, further studies are needed to

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elucidate the function of bld as related to alcohol production.

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3.2. Effects of the overexpression of alcohol dehydrogenase genes on butanol production

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We further attempted to enhance the butanol production in C.

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saccharoperbutylacetonicum through the overexpression of alcohol dehydrogenase genes.

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In C. acetobutylicum, the bi-functional aldehyde/alcohol dehydrogenase encoding genes

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adhE1 and adhE2 were reported to be the key genes responsible for butanol production,

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and adhE1 was identified to play the primary role under solventogenesis (Yoo et al., 2016).

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The overexpression of either adhE1 or its mutant gene adhED485G (the 485th amino acid

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reside Asp was replaced by Gly) has been demonstrated to significantly enhance both

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butanol production and selectivity in C. acetobutylicum (Jang et al., 2012a). There are at

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least two genes, adhE1 (CSPA_C21490) and adhE2 (CSPA_C37180) encoding the

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aldehyde/alcohol dehydrogenase in C. saccharoperbutylacetonicum N1-4. However, due to

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the lack of information concerning their functions for solvent production, we decided to

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overexpress the well characterized adhE1 from C. acetobutylicum ATCC824 as well as its

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mutant adhE1D485G in C. saccharoperbutylacetonicum N1-4, aiming to enhance the butanol

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production.

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Results from the batch fermentation with C. saccharoperbutylacetonicum pSH2, C.

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saccharoperbutylacetonicum pSH3 as well as the control strain C.

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saccharoperbutylacetonicum pTJ1 were illustrated in Fig. 2 and Table 2. Compared to the

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control, both C. saccharoperbutylacetonicum pSH2 and C. saccharoperbutylacetonicum

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pSH3 consumed more glucose (only 3.5 g L-1 and 5.3 g L-1 glucose was left, respectively)

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and reached to a higher OD600 of 13.4. The acetate production (the peak value during the

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fermentation; the same below for all the statement about acid production) in C.

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saccharoperbutylacetonicum pSH2 and C. saccharoperbutylacetonicum pSH3 was similar

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with that in C. saccharoperbutylacetonicum pTJ1, while the butyrate production was

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decreased by 81.3% and 68.8% in C. saccharoperbutylacetonicum pSH2 and C.

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saccharoperbutylacetonicum pSH3, respectively. Both C. saccharoperbutylacetonicum

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pSH2 and C. saccharoperbutylacetonicum pSH3 showed ~14% increase of acetone

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production than the control. In addition, both strains exhibited significantly increased

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ethanol production (5.6- and 4.9-fold higher than the control), resulting in very high ABE

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titers (30.6 and 30.1 g L-1 respectively). These are among the highest total ABE

282

productions ever reported in the batch fermentation with solventogenic clostridia (Grobben

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et al., 1993; Nanda et al., 2017). The overexpression of the mutant adhED485G gene in C.

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saccharoperbutylacetonicum pSH3 led to slight increase (by 0.2 g L-1) in butanol

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production than the overexpression of adhE1 in C. saccharoperbutylacetonicum pSH2.

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However, when compared to the control, the butanol production in both C.

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saccharoperbutylacetonicum pSH2 and C. saccharoperbutylacetonicum pSH3 slightly

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decreased. These results are different from those when the same genes were overexpressed

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in C. acetobutylicum as we described above. This indicated that the alcohol production

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metabolism (particularly the function of the alcohol dehydrogenase in different host

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environment including the flux balance and redox balance) might not be exactly the same

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within different solventogenic clostridia species. The further characterization and

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overexpression of the endogenous alcohol dehydrogenase genes in C.

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saccharoperbutylacetonicum N1-4 might be interesting to evaluate their effects on butanol

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production. On the other hand, when compared with C. saccharoperbutylacetonicum

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pSH2, C. saccharoperbutylacetonicum pSH3 produced less ethanol and meanwhile more

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butanol, confirmed the pervious report that the mutant adhED485G possesses better

13

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cofactor affinities for both NADH and NADPH which is beneficial for enhanced butanol

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production (Jang et al., 2012a).

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Similar as the overexpression of sol operon in C. saccharoperbutylacetonicum pSH1,

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the overexpression of adhE1 in C. saccharoperbutylacetonicum pSH2 and adhED485G in C.

302

saccharoperbutylacetonicum pSH3 enhanced the carbon flow from acetyl-CoA towards

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ethanol production rather than butyryl-CoA production, and thus led to dramatic increase

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in ethanol production but slight decrease in butanol production. Such boost of carbon flux

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accelerated the glucose consumption and cell growth. Although more acetate might have

306

been generated along with the enhanced cell growth, the enhanced flux for acetyl-CoA

307

consumption pulled for more acetate re-assimilation. Thus, no obvious increase of acetate

308

production was observed in either C. saccharoperbutylacetonicum pSH2 or C.

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saccharoperbutylacetonicum pSH3. However, remarkable decrease in butyrate production

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was observed in both recombinant strains, suggesting the promotion of conversion from

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butyryl-CoA to butanol and also the insufficiency of the butyryl-CoA pool due to the

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overexpression of alcohol dehydrogenase genes. Altogether, we concluded that the

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conversion of the carbon flux from acetyl-CoA to butyryl-CoA is the bottleneck in

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improving the butanol production in these recombinant strains. Therefore, in the following

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steps, the genes within the ‘trunk’ pathways responsible for the carbon flux from acetyl-

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CoA to butyryl-CoA were overexpressed and evaluated for butanol production.

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3.3. Effects of the overexpression of thiolase on butanol production

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Thiolase (acetyl-CoA acetyltransferase) converts acetyl-CoA to acetoacetyl-CoA, which

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is the first step for the conversion of acetyl-CoA to butyryl-CoA. The overexpression of

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the thiolase (thl) gene (CA_C2873) in C. acetobutylicum resulted in the decrease of acetate

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and ethanol production along with increased acetone and butyrate production (Sillers et al.,

322

2009). Moreover, the mutant thlAV5A gene of thl (CA_C2873) showed a 32% higher

323

activity than thl. With the overexpression of thlAV5A in C. acetobutylicum ATCC 824, the

324

butanol production was increased by 26% (Cho et al., 2017). There are six thl genes

325

annotated in the genome of C. saccharoperbutylacetonicum N1-4. Except for thl4

326

(CSPA_RS10215) the overexpression of which was reported to hamper ABE fermentation

327

(Herman et al., 2016), there is no report concerning the roles of the other five thl genes.

328

According to the similar arrangement of adjacent genes around thl (CSPA_RS03020) in

329

the genome of C. saccharoperbutylacetonicum N1-4 with that in the genome of C.

330

acetobutylicum ATCC 824, the thl gene (CSPA_RS03020) was predicted as the primary

331

thiolase encoding gene and selected for overexpression in this study. Besides the original

332

thl gene, the mutant gene thlA1V5A was also overexpressed for enhanced butanol production.

333

The thlA1V5A was derived from the thl gene (CSPA_RS03020) based on the similar amino

334

acid alignment between the two genes imitating that between thl (CA_C2873) and thlAV5A.

335

In addition, the thlAV5A (the mutant of thl (CA_C2873)) was also selected to overexpress in

336

C. saccharoperbutylacetonicum here to investigate its effects on butanol production (Cho

337

et al., 2017).

338

As demonstrated in Table 2, recombinant strains overexpressing the thiolase genes

339

reached higher OD600 (at least 13.6% higher than the control strain harboring pTJ1).

340

However, similar glucose consumption kinetics were observed for all these strains as that

341

of the control (Fig. 3A). The overexpression of thiolase genes directed the carbon flux to

342

longer chain metabolites, leading to the reduced production of acetate (0.5, 0.2 and 0.4 g L-

343

1

in C. saccharoperbutylacetonicum N1-4 strains harboring pSH4, pSH5 and pSH6,

15

344

respectively vs. the control) and slight decrease of ethanol (Figs. 3B & 3E, and Table 2).

345

The acetone production in both C. saccharoperbutylacetonicum pSH4 and C.

346

saccharoperbutylacetonicum pSH5 are approximately the same as that in the control.

347

However, a 19.4% decrease in acetone production was observed in C.

348

saccharoperbutylacetonicum pSH6 than the control (Fig. 3D). Unexpectedly, decreased

349

production of butyrate (by 12.5% in C. saccharoperbutylacetonicum pSH5 and 31.3% in

350

both C. saccharoperbutylacetonicum pSH4 and C. saccharoperbutylacetonicum pSH6

351

were observed in all the recombinant strains with the overexpression of different thiolase

352

encoding genes (Fig. 3C). However, the butanol production in C.

353

saccharoperbutylacetonicum pSH4, C. saccharoperbutylacetonicum pSH5 and C.

354

saccharoperbutylacetonicum pSH6 were increased by 0.8 g L-1, 1.3 g L-1 and 0.8 g L-1,

355

respectively compared to the control (Fig. 3F), confirming the positive effects of thiolase

356

overexpression on butanol production.

357

3.4. Effects of the overexpression of expression cassette EC on butanol production

358

As demonstrated above, although the increase of butanol production was observed in all

359

the strains with the overexpression of thiolase genes, such increase is still very limited

360

(from 5.2% to 8.5%). To further drive the carbon flux towards elevated butyryl-CoA

361

production and ultimate butanol production, the whole expression cassette EC including thl,

362

hbd, crt and bcd were overexpressed altogether in C. saccharoperbutylacetonicum pSH7.

363

Fermentation characteristics of C. saccharoperbutylacetonicum pSH7 were illustrated in

364

Fig. 4 and Table 2. It seems like the expression of the large plasmid (about 13 kb) brought

365

some metabolic burden for C. saccharoperbutylacetonicum pSH7, which demonstrated

366

obviously slower glucose consumption and prolonged acid production than C.

16

367

saccharoperbutylacetonicum pSH4 and C. saccharoperbutylacetonicum pTJ1 (Figs. 4A &

368

4B). However, C. saccharoperbutylacetonicum pSH7 reached a much higher OD600 (14.6)

369

than either C. saccharoperbutylacetonicum pSH4 or C. saccharoperbutylacetonicum pTJ1

370

(Table 2). Compared to the control, the acetate production in C.

371

saccharoperbutylacetonicum pSH7 decreased by 8% while the butyrate production was

372

kept the same. When compared to C. saccharoperbutylacetonicum pSH4, a 15% increase

373

in acetate production and a 45.5% increase in butyrate production were observed in C.

374

saccharoperbutylacetonicum pSH7 (Figs. 4B & 4C).

375

With the overexpression of EC, more carbon flux was directed from acetyl-CoA to

376

butyryl-CoA, resulting in dramatic changes in the ABE production profiles (Figs. 4D-F).

377

The acetone production in C. saccharoperbutylacetonicum pSH7 was decreased by 14.9%

378

and 17.4% compared with that in C. saccharoperbutylacetonicum pTJ1 and C.

379

saccharoperbutylacetonicum pSH4, respectively. The ethanol production in C.

380

saccharoperbutylacetonicum pSH7 was also decreased by 58.3% and 54.5% when

381

compared to that in C. saccharoperbutylacetonicum pTJ1 and C.

382

saccharoperbutylacetonicum pSH4, respectively. However, the butanol production in C.

383

saccharoperbutylacetonicum pSH7 reached 17.4 g L-1 with an increase of 2.1 g L-1 and 1.3

384

g L-1 respectively compared with that in C. saccharoperbutylacetonicum pTJ1 and C.

385

saccharoperbutylacetonicum pSH4. With the decrease of acetone and ethanol production

386

and the increase of butanol production, the butanol selectivity was elevated to 73.7%,

387

which is an 11.7% increase compared to the control.

388

In this study, through the overexpression of key genes within the ABE fermentation

389

pathways, especially the genes responsible for driving the carbon flux from acetyl-CoA to

17

390

butyryl-CoA, the butanol production in C. saccharoperbutylacetonicum N1-4 was

391

enhanced. However, the increase of butanol production was still very limited. Much more

392

significant improvement has have been reported in C. acetobutylicum when similar

393

strategies were employed (Hou et al., 2013; Jang et al., 2012a; Lee et al., 2016). On one

394

hand, this suggested that the function of these particular genes as related to butanol

395

production might be different especially in the context of different hosts with different flux

396

and redox status. On the other, C. saccharoperbutylacetonicum N1-4 can naturally produce

397

much higher levels of butanol and ABE than either C. acetobutylicum or C. beijerinckii

398

(Wang et al., 2017); due to the limited tolerance of the host to butanol (and solvents),

399

further massive improvement for butanol production might not be easy to achieve.

400

Therefore, the enhancement of butanol tolerance of the host strain might be right direction

401

to go in order to improve its butanol production capacity. For example, the overexpression

402

of groESL encoding the heat shock proteins was reported to improve cellular tolerances

403

and solvent production in both solventogenic clostridia (Luan et al., 2014; Tomas et al.,

404

2003) and E. coli (Abdelaal et al., 2015). With butanol tolerance considered as one of the

405

major bottlenecks to improve the butanol production, it would be appropriate to

406

overexpress genes enhancing butanol tolerance and the ones driving flux towards butanol

407

production (such as the expression cassette EC as demonstrated in this study) to further

408

improve butanol production in C. saccharoperbutylacetonicum N1-4. Plasmid-based gene

409

overexpression is generally unstable, and usually antibiotics is needed to be added for the

410

fermentation (Ohta et al., 1991), which makes the operation complicated and increases the

411

operational costs. Fortunately, the efficient genome engineering tool has been developed

412

recently for C. saccharoperbutylacetonicum N1-4 in our group (Wang et al., 2017). This

18

413

makes it convenient to integrate the desirable genes into the chromosome along with

414

beneficial clean deletion of genes, thus obtaining metabolically stable mutants. Besides,

415

considering the complicated metabolic pathways and possible metabolic differences among

416

different strains, detailed elucidation of the solvent production mechanism in C.

417

saccharoperbutylacetonicum is necessary through, for example, transcriptomic, proteomic

418

and metabolomic analyses, in order to provide valuable guidance for the metabolic

419

engineering work.

420

4. Conclusions

421

To enhance the butanol production in C. saccharoperbutylacetonicum, key biosynthetic

422

genes including the sol operon (bld-ctfA-ctfB-adc), adhE1, adhE1D485G, thl, thlA1V5A,

423

thlAV5A and expression cassette EC (thl-hbd-crt-bcd) were overexpressed in the host strain.

424

The overexpression of the sol operon increased ethanol production by 400%. The

425

overexpression of adhE1 or the mutant adhE1D485G also resulted in higher ethanol (~5 fold)

426

and total ABE (> 30 g L-1) production. While the most significant increase in butanol

427

production and selectivity were achieved with the overexpression of EC. These results

428

provides a solid foundation for the further development of more robust strains for butanol

429

production.

430 431

Appendix A. Supplementary data

432

E-upplementary data for this work can be found in e-version of this paper online.

433 434 435

19

436

Acknowledgements

437

This work was supported by the Auburn University Intramural Grants Program (IGP), the

438

Hatch program of the USDA National Institute of Food and Agriculture (NIFA), and the

439

USDA-NIFA Southeastern SunGrant. We thank Dr. Hans Blaschek (University of Illinois

440

at Urbana-Champaign) for providing the plasmids.

441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458

20

459

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460

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569 570 571

Figure Captions

25

572

Figure 1. Batch fermentation profiles of the recombinant C. saccharoperbutylacetonicum

573

strains. (A) Glucose consumption; (B) Acetate production; (C) Butyrate production; (D)

574

Acetone production; (E) Ethanol production; (F) Butanol production. pTJ1: C.

575

saccharoperbutylacetonicum pTJ1 harboring the plasmid pTJ1 as the control strain; pSH1:

576

C. saccharoperbutylacetonicum pSH1 harboring the recombinant pSH1 for the

577

overexpression of the sol operon (bld-ctfA-ctfB-adc). Fermentation was carried out in

578

replicates, with one batch reported here as the representative.

579

Figure 2. Batch fermentation profiles of the recombinant C. saccharoperbutylacetonicum

580

strains. (A) Glucose consumption; (B) Acetate production; (C) Butyrate production; (D)

581

Acetone production; (E) Ethanol production; (F) Butanol production. pTJ1: C.

582

saccharoperbutylacetonicum pTJ1 harboring the plasmid pTJ1 as the control strain; pSH2:

583

C. saccharoperbutylacetonicum pSH2 harboring the recombinant plasmid pSH2 for the

584

overexpression of adhE1; pSH3: C. saccharoperbutylacetonicum pSH3 harboring the

585

recombinant pSH3 for the overexpression of the adhE1D485G mutant gene. Fermentation

586

was carried out in replicates, with one batch reported here as the representative. For easy

587

comparison, the same results from the fermentation with C. saccharoperbutylacetonicum

588

pTJ1 as illustrated in Figure 1 are presented here again.

589

Figure 3. Batch fermentation profiles of the recombinant C. saccharoperbutylacetonicum

590

strains. (A) Glucose consumption; (B) Acetate production; (C) Butyrate production; (D)

591

Acetone production; (E) Ethanol production; (F) Butanol production. pTJ1: C.

592

saccharoperbutylacetonicum pTJ1 harboring the plasmid pTJ1 as the control strain; pSH4:

593

C. saccharoperbutylacetonicum pSH4 harboring the recombinant pSH4 for the

594

overexpression of thl (CSPA_RS03020); pSH5: C. saccharoperbutylacetonicum pSH5

26

595

harboring the recombinant plasmid pSH5 for the overexpression of the thlA1V5A mutant

596

gene; pSH6: C. saccharoperbutylacetonicum pSH6 harboring the recombinant pSH6 for

597

the overexpression of the thlAV5A mutant gene. Fermentation was carried out in replicates,

598

with one batch reported here as the representative. For easy comparison, the same results

599

from the fermentation with C. saccharoperbutylacetonicum pTJ1 as illustrated in Figure 1

600

are presented here again.

601

Figure 4. Batch fermentation profiles of the recombinant C. saccharoperbutylacetonicum

602

strains. (A) Glucose consumption; (B) Acetate production; (C) Butyrate production; (D)

603

Acetone production; (E) Ethanol production; (F) Butanol production. pTJ1: C.

604

saccharoperbutylacetonicum pTJ1 harboring the plasmid pTJ1 as the control strain; pSH4:

605

C. saccharoperbutylacetonicum pSH4 harboring the recombinant pSH4 for the

606

overexpression of thl (CSPA_RS03020); pSH7: C. saccharoperbutylacetonicum pSH7

607

harboring the recombinant pSH7 for the overexpression of the expression cassette EC (thl-

608

hbd-crt-bcd). Fermentation was carried out in replicates, with one batch reported here as

609

the representative. For easy comparison, the same results from the fermentation with C.

610

saccharoperbutylacetonicum pTJ1 as illustrated in Figure 1 and with C.

611

saccharoperbutylacetonicum pSH4 as illustrated in Figure 3 are presented here again.

612 613 614 615 616 617 618

Table 1

27

619

Strains and plasmids used in this study Name Strains C. saccharoperbutylacetonicum N1-4 E. coli ER2523 (NEB Express)

Characteristics

Sources or references

DSM 14923 (= ATCC 27021) fhuA2 [lon] ompT gal sulA11 R(mcr73::miniTn10--TetS)2 [dcm] R(zgb210::Tn10--TetS) endA1 Δ(mcrCmrr)114::IS10

DSM New England Biolabs

C. acetobutylicum ATCC 824 Plasmids (mother vectors) pTJ1 pYW25 Recombinant plasmids pSH1

pSH2 pSH3 pSH4 pSH5 pSH6 pSH7

ATCC CAK1 ori Ampr Ermr CAK1 ori Ampr Ermr::Pthl Overexpressed gene bld (CSPA_RS27680) ctfA (CSPA_RS27685) ctfB (CSPA_RS27690) adc (CSPA_RS27695) adhE1 (CA_P0162) adhE1D485G(CA_P0162) thl (CSPA_RS03020) thlA1V5A (CSPA_RS03020) thlAV5A (CAC2873) thl (CSPA_RS03020) hbd (CSPA_RS02150) crt (CSPA_RS2130) bcd (CSPA_RS2150)

620 621 622 623 624 625 626 627 628 629 630 631 632

Table 2

28

(Wang et al., 2013) Lab stock This study

This study This study This study This study This study This study

633

Summary of the fermentation results with various C. saccharoperbutylacetonicum strains. Characteristics a Maximum OD600 Peak acetate (g L-1) b Peak butyrate ( g L-1) Butanol (g L-1) c Butanol yield (g g-1) Acetone ( g L-1) Ethanol ( g L-1) Total ABE ( g L-1) Butanol selectivity (g g-1) d

pTJ1 11.8±0.2 2.5±0.1 1.6±0.0 15.3±0.1 0.24±0.0 6.7±0.1 1.2±0.0 23.2±0.1 66.0%±0. 1%

pSH1 12.8±0.2 1.7±0.0 0.1±0.0 12.6±0.1 0.21±0.0 5.7±0.1 6.0±0.0 24.3±0.1 51.9%±0. 1%

pSH2 13.4±0.3 2.4±0.0 0.3±0.0 15.0±0.1 0.19±0.0 7.7±0.1 7.9±0.1 30.6±0.1 49.0%±0. 3%

pSH3 13.4±0.1 2.6±0.1 0.5±0.0 15.2±0.1 0.20±0.0 7.8±0.1 7.1±0.1 30.1±0.1 50.5%±0. 2%

pSH4 13.6±0.1 2.0±0.0 1.1±0.0 16.1±0. 0.25±0.0 6.9±0.1 1.1±0.0 24.1±0.1 66.8%±0. 2%

pSH5 13.4±0.1 2.3±0.1 1.4±0.0 16.6±0.0 0.26±0.0 6.5±0.0 1.0±0.0 24.1±0.0 68.9%±0. 1%

pSH6 14.2±0.2 2.1±0.0 1.1±0.0 16.1±0.0 0.27±0.0 5.4±0.1 1.1±0.0 22.6±0.01 71.2%±0. 1%

634

a

635

saccharoperbutylacetonicum pTJ1, etc.

636

b

There was approximately 1.7 g l-1 of acetate pre-added in the P2 medium.

637

c

The reported titers were the maximum values after the solvent production reached plateaus.

638

d

Butanol selectivity: the ratio of butanol out of the total ABE.

pSH7 14.6±0.2 2.3±0.1 1.6±0.1 17.4±0.0 0.27±0.0 5.7±0.1 0.5±0.0 23.6±0.0 73.7%±0. 1%

The plasmid is used to represent the corresponding strain that contains the plasmid. For example, pTJ1 = C.

639

29

640 641

30

642 643

31

644 645

32

646 647

33

648

Highlights

649



Overexpression of sol operon enhanced acid re-assimilation and ethanol titer.

650



Overexpression of adhE increased ethanol and total ABE titer dramatically.

651



Overexpression of thl decreased ethanol and increased butanol production slightly.

652



Overexpression of cassette EC improved butanol titer and selectivity.

653

34