Recruiting Energy-Conserving Sucrose Utilization Pathways for

Letter pubs.acs.org/journal/ascecg

Recruiting Energy-Conserving Sucrose Utilization Pathways for Enhanced 2,3-Butanediol Production in Bacillus subtilis Jun Feng,†,‡ Yanyan Gu,† Peng-Fei Yan,† Cunjiang Song,§ and Yi Wang*,†,∥ †

Department of Biosystems Engineering, Auburn University, Auburn, Alabama 36849, United States Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, Shaanxi 710049, China § Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, Nankai University, Tianjin 300071, China ∥ Center for Bioenergy and Bioproducts, Auburn University, Auburn, Alabama 36849, United States ‡

S Supporting Information *

ABSTRACT: To improve the utilization of sucrose for 2,3-butanediol (2,3-BD) production, four combinations of heterologous energy-conserving sucrose utilization pathways were introduced into Bacillus subtilis Δtet strain (a derivative from B. subtilis 168) and B. subtilis FJ-1 strain (a new isolate in our lab). Results demonstrated that the combination of cscB (encoding sucrose permease) from Escherichia coli and gtfA (encoding sucrose phosphorylase) from Streptococcus mutans showed the most remarkable enhancement for the 2,3-BD production. With sucrose and sugar cane juice as substrate, respectively, the Δtet-CEG strain (B. subtilis Δtet strain containing the energy-conserving sucrose utilization pathway as referred above) showed 36.5% and 24.7% increase in 2,3-BD production than the control Δtet strain, and the FJ-1-CEG strain also produced 23.8% and 44.5% more 2,3-BD than the control FJ-1 strain. The metabolic engineering strategy demonstrated in this study can be extensively applied to other microorganisms for reinforced production of desirable biochemicals from sucrose. Meanwhile, the newly isolated FJ-1 strain is an interesting platform strain that can be further engineered for efficient 2,3-BD production. KEYWORDS: Bacillus subtilis, Sucrose, Energy-conserving pathways, 2,3-Butanediol

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INTRODUCTION

for efficient 2,3-BD production, and would be preferable options for safe industrial fermentations.4 Various metabolic engineering strategies have been implemented to improve 2,3-BD production in the host microorganisms, such as the overexpression of genes related to the 2,3-BD synthesis,5 the enhancement of intracellular NADH levels,3 and the deletion of genes for byproduct production.6 However, until now, there are few reports focusing on

2,3-butanediol (2,3-BD) is a valuable platform chemical, which has potential applications as feedstock for the production of pharmaceuticals, foods, cosmetics, fuel additives, and etc.1,2 Currently, the industrial production of 2,3-BD is mostly through the chemical synthesis routes, which are capitally costly and nonenvironmentally friendly. As an alternative, the production of 2,3-BD through microbial fermentation is attracting more and more interests because it is generally less expensive and more environmentally friendly.3 Bacillus subtilis, which is generally regarded as safe (GRAS), can be employed © 2017 American Chemical Society

Received: October 8, 2017 Revised: October 24, 2017 Published: November 1, 2017 11221

DOI: 10.1021/acssuschemeng.7b03636 ACS Sustainable Chem. Eng. 2017, 5, 11221−11225

Letter

ACS Sustainable Chemistry & Engineering

Figure 1. Sucrose utilization pathways. (a) The sucrose utilization pathway consists of PTS and sucrose-6-P hydrolase; (b) non-PTS sucrose utilization pathway consists of sucrose permease and sucrose phosphorylase. Texts in blue represent the introduced heterologous energy-conserving sucrose utilization pathways. The native system for sucorse utilization of these two B. subtilis strains is PTS assoicated sucrose utilization pathway.

reinforcing 2,3-BD production through metabolic engineering to boost the carbon source utilization. In microorganisms, carbon metabolic pathways are responsible for the generation of ATP, balancing of redox-cofactors, and the synthesis of precursors. Enhancement of the carbon metabolism pathways is a very reasonable strategy to improve the production of the desirable endproducts. Sucrose is one of the most abundant and least expensive carbon sources that can be easily obtained from, for example, sugar cane, and sugar beet. Recently, the attempts for utilization of sucrose-based feedstock for various biofuel and biochemicals production through microbial fermentation have attracted great attention. The sucrose-based feedstock has been widely used for the production of ethanol, n-butanol, biodiesel, and etc.7−9 Compared to these bioproducts, 2,3-BD has a smaller global market but has much higher value as a biochemical.10 It has been reported that the potential global market for 2,3-BD is about 32 million tons per annum which values nearly $43 billion.10,11 Thus, engineering the sucrose metabolic pathway in B. subtilis is a promising strategy to improve the 2,3-BD production from low-value sucrose-based feedstocks. The sucrose utilization pathway in B. subtilis is based on a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) (Figure 1a).12 Through this pathway, two molecules of ATP are required for the conversion of one molecule sucrose into fructose-6-P and glucose-6-P (the consumption of one molecule PEP corresponds to the consumption of one molecule ATP). On the other hand, there is a non-PTS sucrose utilization pathway existing in nature which is considered to be an energy-conserving one (Figure 1b).13 Compared to the PTS pathway showed in Figure 1a, only one molecule of ATP will be used through this pathway for the conversion of one molecule sucrose into fructose-6-P and glucose-6-P.

In this study, we aimed to enhance the sucrose utilization metabolism by introducing the energy-conserving pathways into B. subtilis strains for improved 2,3-BD production. Four heterologous energy-conserving sucrose utilization pathways were introduced into the B. subtilis Δtet strain and FJ-1 strain (a new isolate in our lab) to evaluate their effects on 2,3-BD production in the host. Results indicated that both of the final 2,3-BD production and yield were significantly increased in the appropriate recombinant strains. To our best knowledge, this is the first report concerning the increase of microbial 2,3-BD production by introducing energy-conserving sucrose utilization pathways. Such a strategy can be extensively applied to other microorganisms for reinforced production of desirable biochemicals from sucrose.

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EXPERIMENTAL SECTION

Microorganisms and Cultivation Conditions. All the strains and plasmids used in this study are listed in Table S1. B. subtilis N-3, N-5, N-6, A-1, FJ-1 and FJ-2 strains were newly isolated from fermented foods (Chinese natto and sufu from the local grocery store) as described previously.14 2,3-BD fermentations were carried out using the 2,3-BD fermentation medium (containing 117.3 g/L sucrose; 15 g/L yeast extract; 3 g/L tryptone; 3 g/L NaCl; 3 g/L (NH4)2SO4; 0.6 g/L MgSO4; 1 mM FeSO4; 1 mM CaCl2; 1 mM MnSO4 and 1 mM ZnCl2) in 250 mL shaking flasks at 37 °C and 150 rpm for 120 h. When sugar cane juice was used as the feedstock, two times diluted sugar cane juice (contains 91.7 g/L sucrose, 11.9 g/L glucose, 8.9 g/L fructose) was used to replace the sucrose in the 2,3-BD fermentation medium. Construction of Recombinant Strains. To delete the tetL gene, a marker-less gene deletion method as described previously was employed.15,16 The generated mutant was designated as B. subtilis Δtet. The primers used for deletion in this study are listed in Table S2. Energy-saving sucrose consumption pathways contain two enzymes: sucrose permease and sucrose phosphorylase. We have previously constructed four combinations of the energy-saving sucrose consumption pathways and the plasmids carrying these genes were 11222


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ACS Sustainable Chemistry & Engineering designated as pWH1520-CES, pWH1520-CEG, pWH1520-CBS, and pWH1520-CBG, respectively (Table S1). The plasmids were transformed into the target strains according to a previously described method.13 Analytical Procedures. The concentration of 2,3-BD was measured using an HPLC (Agilent Technologies 1260 Infinity series, CA) equipped with a refractive index Detector (RID) and a diode array ultraviolet detector (DAD), along with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA). The column was eluted by 0.005 mol/L H2SO4 with a flow rate of 0.6 mL/min at 65 °C. Concentrations of sucrose, glucose and fructose were measured using an HPLC with an Aminex HPX-87P column. The column was eluted by ddH2O with a flow rate of 0.6 mL/min at 35 °C.

(cscB (B. lactis), CAD26969.1) and two sucrose phosphorylase d e r i v e d f r o m B ifi d ob a c t e r i u m a d o l e s c e n t i s ( s u c P , WP_011742626.1) and Streptococcus mutans (gtfA, WP_002262875.1) were recruited to construct the heterogeneous energy-saving sucrose utilization pathway (Table 1).18 Table 1. Heterologous Energy-Conserving Sucrose Utilization Pathways Introduced into Bacillus subtilis Strains for Enhanced 2,3-Butanediol Production18 pathways with combinations of genes

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CES

RESULTS AND DISCUSSION Six strains (N-3, N-5, N-6, A-1, FJ-1, and FJ-2) were isolated from fermented foods. They were all confirmed as B. subtilis strains through the analysis of their 16S rDNA gene sequence (data not shown).14 As a starting point, first, the six newly isolated strains along with the 168 strain (the type strain of B. subtilis) were evaluated for their capability for 2,3-BD production through fermentation using sucrose as the feedstock. As shown in Figure 2, all the new isolates can produce

CEG CBS CBG

gene source cscB from Escherichia coli and sucP from Bifidobacterium adolescentis cscB from Escherichia coli and gtfA from Streptococcus mutans cscB from Bifidobacterium lactis and sucP from Bifidobacterium adolescentis cscB from Bifidobacterium lactis and gtfA from Streptococcus mutans

The four heterologous energy-conserving sucrose utilization pathways were introduced into the B. subtilis Δtet strain and FJ1 strain to evaluate their effects on 2,3-BD production in the host. The fermentation results indicated that the new pathways can not only increase the sucrose consumption but also improve the 2,3-BD production (Figure 3). Among them, the CEG pathway (combination of cscB from E. coli and gtfA from S. mutans) demonstrated the most significant effects for both strains (Δtet-CEG and FJ-1-CEG). The Δtet-CEG strain produced 24.6 g/L of 2,3-BD, which was 36.5% higher than that of the control Δtet strain (18.0 g/L). The FJ-1-CEG strain produced 44.6 g/L of 2,3-BD, which was 23.8% higher than that of the control FJ-1 strain (36.0 g/L). These two mutant strains also showed the highest yield of 2,3-BD. The Δtet-CEG strain exhibited a yield of 0.35 g/g, which was 25.0% higher than that of the control Δtet strain. The FJ-1-CEG strain exhibited a yield of 0.45 g/g, which was 15.4% higher than that of the control FJ-1 strain. This yield is about 90% of the theoretical maximum yield. Further fermentations were carried out with recombinant strains using sugar cane juice as the carbon source. As shown in Figure 4, the Δtet-CEG strain produced 27.0 g/L 2,3-BD with a yield of 0.28 g/g. The titer and yield were 24.7% and 21.7% higher than those of the Δtet strain, respectively. On the other hand, the FJ-1-CEG strain generated 34.2 g/L 2,3-BD with a yield of 0.32 g/g. These were 44.5% (for the titer) and 39.1% (for the yield) higher than that of the FJ-1 strain, respectively. These results indicated that the energy-conserving sucrose utilization pathways could also enhance the 2,3-BD production and yield when sugar cane juice was used as the substrate. However, interestingly, compared to the fermentation with the sucrose as the substrate, the 2,3-BD production from sugar cane juice in Δtet and Δtet-CEG strains was both slightly increased, whereas that in the FJ-1 and FJ-1-CEG strains was dramatically decreased (by 34.2% and 23.3% for FJ-1 and FJ-1-CEG, respectively). We reasoned that the impurity components in the sugar cane juice might have led to inhibitory effects on the 2,3-BD production in FJ-1 and its derived strains. The B. subtilis 168 was obtained from its ancestor (which was originally isolated from soil environment) and underwent domestication processes of mutagenesis and selection,19,20 whereas the FJ-1 strain was isolated from the high saline environment in the fermented food. Comparatively, B. subtilis 168 and its derivative

Figure 2. 2,3-butanediol fermentation results using sucrose as the substrate with Bacillus subtilis N-3, N-5, N-6, A-1, FJ-1, FJ-2, and 168 strains. Values represent means ± SD of triplicates.

comparable or higher 2,3-BD from sucrose than the 168 strain. The 168 strain generated 18.5 g/L of 2,3-BD. For the six new isolates, the A-1 strain produced the lowest concentration of 2,3-BD (15.6 g/L) and the FJ-1 strain produced the highest concentration of 2,3-BD (35.5 g/L). Therefore, we selected the FJ-1 strain along with the type strain (168 strain) for further genetic manipulation for enhanced sucrose consumption and 2,3-BD production. The plasmid pWH1520 (the mother vector for introducing the energy-conserving sucrose utilization pathways) harbors a tetracycline resistance gene for selection. Our preliminary experiments showed that FJ-1 strain was sensitive to tetracycline while B. subtilis 168 was resistant to tetracycline because it contains a tetracycline efflux MFS transporter (TetL) on the chromosome.4 Thus, in order to transform the pWH1520 derived plasmids into the 168 strain, the tetL gene must be deleted.17 To delete the tetL gene, a marker-less gene deletion method as described previously was employed.15,16 The derived strain with the tetL gene deleted was desinaged as B. subtilis Δtet. The heterologous energy-saving sucrose consumption pathways contain two enzymes: sucrose permease and sucrose phosphorylase. Two sucrose permeases derived from E. coli W (cscB (E. coli), WP_001197025.1) and Bifidobacterium lactis 11223


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Figure 3. 2,3-butanediol (2,3-BD) fermentation results with sucrose as the substrate. (a) 2,3-BD production in Δtet, Δtet-CES, Δtet-CEG, Δtet-CBS, and Δtet-CBG strains; (b) sucrose consumption in Δtet, Δtet-CES, Δtet-CEG, Δtet-CBS, and Δtet-CBG strains; (c) 2,3-BD production in FJ-1, FJ-1CES, FJ-1-CEG, FJ-1-CBS, and FJ-1-CBG strains; (d) sucrose consumption in FJ-1, FJ-1-CES, FJ-1-CEG, FJ-1-CBS, and FJ-1-CBG strains. All the fermentations were carried out with the 2,3-BD fermentation medium. Samples were taken at 120 h for the measurement of the 2,3-BD production and sugar consumption. Values represent means ± SD of triplicates.

Figure 4. 2,3-butanediol (2,3-BD) fermentation results with sugar cane juice as the substrate. (a) 2,3-BD production in Δtet, Δtet-CEG, FJ-1, and FJ1-CEG strains; (b) sugar consumption in Δtet, Δtet-CEG, FJ-1, and FJ-1-CEG strains. Samples were taken at 120 h for the measurement of the 2,3BD production and sugar consumption. Values represent means ± SD of triplicates.

ATP supply. The introduction of energy-conserving sucrose utilization pathways saves energy (ATP) for the regular cell growth, and therefore saves pyruvate for 2,3-BD production. Interestingly, compared to other combinations of pathways, the CEG combination resulted in the least increase in carbon consumption but the highest increase in 2,3-BD titer and yield (Figure 3). The sucrose permeases and sucrose phosphorylases tested here are from different microorganisms, and thus they are unlikely to be fully functional in the new hosts due to the heterologous regulation machineries. Therefore, the optimized combination of the two enzymes is very important for the appropriate function of the whole pathway. Results from this study suggested that the CEG pathway represents the most

strains are more adaptable to the sugar cane juice, and actually the additional nutrients (besides sucrose) might have benefited the cell growth and 2,3-BD production, whereas FJ-1 and derivative strains have suffered the inhibition from the impurities in the sugar cane juice. The introduction of the energy-conserving sucrose utilization pathway has led to boosted sugar consumption and 2,3-BD production in both the B. subtilis type strain and the new isolate. Two possible benefits could contribute to this outcome: (1) recruiting another sucrose utilization pathway enhanced the carbon source utilization in the host strain, and thus improved the synthesis of the desirable product; (2) pyruvate is the most important precursor for TCA cycle and 2,3-BD synthesis. Meanwhile, TCA cycle is the primary pathway for intracellular 11224


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redistributing the carbon flux to 2, 3-butanediol by manipulating NADH levels. Biotechnol. Biofuels 2015, 8, 129. (4) Fu, J.; Huo, G.; Feng, L.; Mao, Y.; Wang, Z.; Ma, H.; Chen, T.; Zhao, X. Metabolic engineering of Bacillus subtilis for chiral pure meso2, 3-butanediol production. Biotechnol. Biofuels 2016, 9, 90. (5) Kim, B.; Lee, S.; Park, J.; Lu, M.; Oh, M.; Kim, Y.; Lee, J. Enhanced 2, 3-butanediol production in recombinant Klebsiella pneumoniae via overexpression of synthesis-related genes. J. Microbiol. Biotechnol. 2012, 22, 1258−1263. (6) Li, Z.-J.; Jian, J.; Wei, X.-X.; Shen, X.-W.; Chen, G.-Q. Microbial production of meso-2, 3-butanediol by metabolically engineered Escherichia coli under low oxygen condition. Appl. Microbiol. Biotechnol. 2010, 87, 2001−2009. (7) Moraes, B. S.; Zaiat, M.; Bonomi, A. Anaerobic digestion of vinasse from sugarcane ethanol production in Brazil: Challenges and perspectives. Renewable Sustainable Energy Rev. 2015, 44, 888−903. (8) Soccol, C. R.; Neto, C. J. D.; Soccol, V. T.; Sydney, E. B.; da Costa, E. S. F.; Medeiros, A. B. P.; de Souza Vandenberghe, L. P. Pilot scale biodiesel production from microbial oil of Rhodosporidium toruloides DEBB 5533 using sugarcane juice: performance in diesel engine and preliminary economic study. Bioresour. Technol. 2017, 223, 259−268. (9) Zhang, J.; Yu, L.; Lin, M.; Yan, Q.; Yang, S.-T. n-Butanol production from sucrose and sugarcane juice by engineered Clostridium tyrobutyricum overexpressing sucrose catabolism genes and adhE2. Bioresour. Technol. 2017, 233, 51−57. (10) Köpke, M.; Mihalcea, C.; Liew, F.; Tizard, J. H.; Ali, M. S.; Conolly, J. J.; Al-Sinawi, B.; Simpson, S. D. 2, 3-Butanediol production by acetogenic bacteria, an alternative route to chemical synthesis, using industrial waste gas. Appl. Environ. Microbiol. 2011, 77, 5467−5475. (11) Li, L.; Zhang, L.; Li, K.; Wang, Y.; Gao, C.; Han, B.; Ma, C.; Xu, P. A newly isolated Bacillus licheniformis strain thermophilically produces 2, 3-butanediol, a platform and fuel bio-chemical. Biotechnol. Biofuels 2013, 6, 123. (12) Postma, P.; Lengeler, J.; Jacobson, G. Phosphoenolpyruvate: carbohydrate phosphotransferase systems of bacteria. Microbiol. Rev. 1993, 57, 543−594. (13) Kitaoka, M.; Hayashi, K. Carbohydrate-processing phosphorolytic enzymes. Trends Glycosci. Glycotechnol. 2002, 14, 35−50. (14) Yan, P.-F.; Feng, J.; Dong, S.; Wang, M.; Khan, I. A.; Wang, Y. Production of high levels of chirally pure d-2, 3-Butanediol with a newly isolated Bacillus strain. ACS Sustainable Chem. Eng. 2017, DOI: 10.1021/acssuschemeng.7b02910. (15) Zhang, W.; Gao, W.; Feng, J.; Zhang, C.; He, Y.; Cao, M.; Li, Q.; Sun, Y.; Yang, C.; Song, C.; Wang, S. A markerless gene replacement method for B. amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-γ-glutamic acid production. Appl. Microbiol. Biotechnol. 2014, 98, 8963−8973. (16) Feng, J.; Gu, Y.; Quan, Y.; Cao, M.; Gao, W.; Zhang, W.; Wang, S.; Yang, C.; Song, C. Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering. Metab. Eng. 2015, 32, 106−115. (17) Sakaguchi, R.; Shishido, K. Molecular cloning of a tetracyclineresistance determinant from Bacillus subtilis chromosomal DNA and its expression in Escherichia coli and B. subtilis. Biochim. Biophys. Acta, Gene Struct. Expression 1988, 949, 49−57. (18) Feng, J.; Gu, Y.; Quan, Y.; Gao, W.; Dang, Y.; Cao, M.; Lu, X.; Wang, Y.; Song, C.; Wang, S. Construction of energy-conserving sucrose utilization pathways for improving poly-γ-glutamic acid production in Bacillus amyloliquefaciens. Microb. Cell Fact. 2017, 16, 98. (19) Burkholder, P. R.; Giles, N. H., Jr. Induced biochemical mutations in Bacillus subtilis. Am. J. Bot. 1947, 34, 345−348. (20) Zeigler, D. R.; Prágai, Z.; Rodriguez, S.; Chevreux, B.; Muffler, A.; Albert, T.; Bai, R.; Wyss, M.; Perkins, J. B. The origins of 168, W23, and other Bacillus subtilis legacy strains. J. Bacteriol. 2008, 190, 6983− 6995.

efficient combination for enhanced sucrose metabolism and 2,3-BD production in B. subtilis. To conclude, the sucrose metabolism in B. subtilis was enhanced by introducing heterologous energy-conserving sucrose utilization pathways in this study. All the four introduced pathways reinforced the sucrose consumption and 2,3-BD production in B. subtilis Δtet and FJ-1 strains. Among them, the CEG pathway with the combination of cscB from E. coli and gtfA from S. mutans demonstrated the most significant positive effects when either sucrose or sugar cane juice was used as the substrate. To our best knowledge, this is the first report concerning the increase of microbial 2,3-BD production by introducing energy-conserving sucrose utilization pathways. Such strategy can be extensively applied to other microorganisms for reinforced production of desirable biochemicals from sucrose.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b03636. Strains, plasmids, and primers used in this work (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Yi Wang, Tel: 1-334-844-3503; Fax: 1-334-844-3530; E-mail: [email protected]. ORCID

Yi Wang: 0000-0002-0192-3195 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Auburn University Intramural Grants Program (IGP), and the Hatch program of the United States Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA). Jun Feng is a postdoc research fellow supported by the Postdoc fellowship from the Auburn University Hatch program of the USDA-NIFA. We thank Dr. Hardev Sandhu (University of Florida) for providing the sugar cane juice.

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ABBREVIATIONS 2,3-BD:2,3-butanediol; ATP:adenosine triphosphate; cscB:sucrose permease gene; gtfA:sucrose phosphorylase gene from Streptococcus mutans; NADH:nicotinamide adenine dinucleotide; PEP:phosphoenolpyruvate; PTS:phosphotransferase system; sucP:sucrose phosphorylase gene from Bifidobacterium adolescentis; TCA:tricarboxylic acid; tetL:gene encoding tetracycline efflux MFS transporter

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REFERENCES

(1) Ji, X.-J.; Huang, H.; Ouyang, P.-K. Microbial 2, 3-butanediol production: a state-of-the-art review. Biotechnol. Adv. 2011, 29, 351− 364. (2) Białkowska, A. M. Strategies for efficient and economical 2, 3butanediol production: new trends in this field. World J. Microbiol. Biotechnol. 2016, 32, 200. (3) Yang, T.; Rao, Z.; Hu, G.; Zhang, X.; Liu, M.; Dai, Y.; Xu, M.; Xu, Z.; Yang, S.-T. Metabolic engineering of Bacillus subtilis for 11225


Recruiting Energy-Conserving Sucrose Utilization Pathways for

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