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Bio-production of high-purity propionate by engineering l-threonine degradation pathway in Pseudomonas putida

  • Biotechnological products and process engineering
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Abstract

Propionic acid (PA) is widely used in the food, agricultural, and pharmaceutical industries. Since the petrochemical PA is unsustainable, biological production of PA from renewable substrates is gaining attention. In this study, we engineered the strain Pseudomonas putida KT2440 to transform l-threonine to PA with only CO2 released as by-product. The cell factory was created by chromosomal incorporation of heterologous l-threonine deaminase, permease, and acyl-CoA thioesterase, deletion of branch pathways as well as overproduction of the endogenous branched-chain alpha-keto acid dehydrogenase complex. The final engineered strain could produce 399 mM PA from 400 mM l-threonine in a batch biotransformation process, with a molar yield of 99.8% under the optimized conditions in 48 h. The PA titer further reached to 50.3 g/L (679 mM) with a productivity of 0.6 g/L/h in a fed-batch conversion process. No obvious by-products, such as acetate and succinate, were detected in the broth, which would significantly facilitate downstream purification steps. Thus, this study offers an alternative route for biological production of PA.

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References

  • Aevarsson A, Chuang JL, Wynn RM, Turley S, Chuang DT, Hol WGJ (2000) Crystal structure of human branched-chain alpha-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease. Structure 8:277–291

    Article  CAS  PubMed  Google Scholar 

  • Akawi L, Srirangan K, Liu X, Moo-Young M, Chou CP (2015) Engineering Escherichia coli for high-level production of propionate. J Ind Microbiol Biotechnol 42:1057–1072

    Article  CAS  PubMed  Google Scholar 

  • Bauerle RH, Freundlich M, Umbarger HE, Stormer FC (1964) Control of isoleucine, valine and leucine biosynthesis: II. Endoproduct inhibition by valine of acetohydroxy acid synthetase in Salmonella typhimurium. Biochim Biophys Acta 92:142–149

    CAS  PubMed  Google Scholar 

  • Belda E, van Heck RGA, Lopez-Sanchez MJ, Cruveiller S, Barbe V, Fraser C, Klenk HP, Petersen J, Morgat A, Nikel PI, Vallenet D, Rouy Z, Sekowska A, dos Santos VAPM, de Lorenzo V, Danchin A, Medigue C (2016) The revisited genome of Pseudomonas putida KT2440 enlightens its value as a robust metabolic chassis. Environ Microbiol 18:3403–3424

    Article  CAS  PubMed  Google Scholar 

  • Calero P, Jensen SI, Bojanovic K, Lennen RM, Koza A, Nielsen AT (2018) Genome-wide identification of tolerance mechanisms toward p-coumaric acid in Pseudomonas putida. Biotechnol Bioeng 115:762–774

  • Casini A, MacDonald JT, de Jonghe J, Christodoulou G, Freemont PS, Baldwin GS, Ellis T (2014) One-pot DNA construction for synthetic biology: the modular overlap-directed assembly with linkers (MODAL) strategy. Nucleic Acids Res 42:e7

    Article  CAS  PubMed  Google Scholar 

  • Chen Y, Wang T, Shen N, Zhang F, Zeng RJ (2016) High-purity propionate production from glycerol in mixed culture fermentation. Bioresour Technol 219:659–667

    Article  CAS  PubMed  Google Scholar 

  • Cho JH, Kim EK, So JS (1995) Improved transformation of Pseudomonas putida KT2440 by electroporation. Biotechnol Tech 9:41–44

    Article  CAS  Google Scholar 

  • Choi KR, Lee SY (2019) Protocols for RecET-based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida. Microb Biotechnol 13:199–209. https://doi.org/10.1111/1751-7915.13374

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Choi KR, Jang WD, Yang D, Cho JS, Park D, Lee SY (2019) Systems metabolic engineering strategies: integrating systems and synthetic biology with metabolic engineering. Trends Biotechnol 37:817–837

    Article  CAS  PubMed  Google Scholar 

  • Cui Q, Zhou F, Liu W, Tao Y (2017) Avermectin biosynthesis: stable functional expression of branched chain alpha-keto acid dehydrogenase complex from Streptomyces avermitilis in Escherichia coli by selectively regulating individual subunit gene expression. Biotechnol Lett 39:1567–1574

    Article  CAS  PubMed  Google Scholar 

  • Dishisha T, Stahl A, Lundmark S, Hatti-Kaul R (2013) An economical biorefinery process for propionic acid production from glycerol and potato juice using high cell density fermentation. Bioresour Technol 135:504–512

    Article  CAS  PubMed  Google Scholar 

  • Dong X, Quinn PJ, Wang X (2011) Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for the production of L-threonine. Biotechnol Adv 29:11–23

    Article  CAS  PubMed  Google Scholar 

  • Elmore JR, Furches A, Wolff GN, Gorday K, Guss AM (2017) Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440. Metab Eng Commun 5:1–8

    Article  PubMed  PubMed Central  Google Scholar 

  • Feng XH, Chen F, Xu H, Wu B, Yao J, Ying HJ, Ouyang PK (2010) Propionic acid fermentation by Propionibacterium freudenreichii CCTCC M207015 in a multi-point fibrous-bed bioreactor. Bioprocess Biosyst Eng 33:1077–1085

    Article  CAS  PubMed  Google Scholar 

  • Feng XH, Chen F, Xu H, Wu B, Li H, Li S, Ouyang PK (2011) Green and economical production of propionic acid by Propionibacterium freudenreichii CCTCC M207015 in plant fibrous-bed bioreactor. Bioresour Technol 102:6141–6146

    Article  CAS  PubMed  Google Scholar 

  • Gonzalez-Garcia RA, McCubbin T, Navone L, Stowers C, Nielsen LK, Marcellin E (2017) Microbial propionic acid production. Fermentation 3:21

    Article  CAS  Google Scholar 

  • Goss TJ, Schweizer HP, Datta P (1988) Molecular characterization of the tdc operon of Escherichia coli K-12. J Bacteriol 170:5352–5359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hesslinger C, Fairhurst SA, Sawers G (1998) Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate. Mol Microbiol 27:477–492

    Article  CAS  PubMed  Google Scholar 

  • Horswill AR, Escalante-Semerena JC (1999) The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase. Microbiology 145:1381–1388

    Article  CAS  PubMed  Google Scholar 

  • Kagan ZS, Dorozhko AI, Kovaleva SV, Yakovleva LI (1975) Studies of homogeneous “biosynthetic” l-threonine dehydratase from Escherichia coli K-12: some kinetic properties and molecular multiplicity. Biochim Biophys Acta 403:208–220

    Article  CAS  PubMed  Google Scholar 

  • Li R, Wijma HJ, Song L, Cui Y, Otzen M, Tian Y, Du J, Li T, Niu D, Chen Y, Feng J, Han J, Chen H, Tao Y, Janssen DB, Wu B (2018) Computational redesign of enzymes for regio- and enantioselective hydroamination. Nat Chem Biol 14:664–670

    Article  CAS  PubMed  Google Scholar 

  • Liu JQ, Dairi T, Itoh N, Kataoka M, Shimizu S, Yamada H (1998) Gene cloning, biochemical characterization and physiological role of a thermostable low-specificity L-threonine aldolase from Escherichia coli. Eur J Biochem 255:220–226

    Article  CAS  PubMed  Google Scholar 

  • Nelson KE (2002) The complete genome sequence of Pseudomonas putida KT2440 is finally available - foreword. Environ Microbiol 4:777–778

    Article  Google Scholar 

  • Nikel PI, de Lorenzo V (2018) Pseudomonas putida as a functional chassis for industrial biocatalysis: from native biochemistry to trans-metabolism. Metab Eng 50:142–155

    Article  CAS  PubMed  Google Scholar 

  • Palacios S, Starai VJ, Escalante-Semerena JC (2003) Propionyl coenzyme A is a common intermediate in the 1,2-propanediol and propionate catabolic pathways needed for expression of the prpBCDE operon during growth of Salmonella enterica on 1,2-propanediol. J Bacteriol 185:2802–2810

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Poblete-Castro I, Becker J, Dohnt K, dos Santos VM, Wittmann C (2012) Industrial biotechnology of Pseudomonas putida and related species. Appl Microbiol Biotechnol 93:2279–2290

    Article  CAS  PubMed  Google Scholar 

  • Quenee L, Lamotte D, Polack B (2005) Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in Pseudomonas aeruginosa. Biotechniques 38:63–67

    Article  CAS  PubMed  Google Scholar 

  • Schweizer HP (1991) Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene 97:109–112

    Article  CAS  PubMed  Google Scholar 

  • Schweizer HP, Hoang TT (1995) An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15–22

    Article  CAS  PubMed  Google Scholar 

  • Simon R, Priefer U, Puhler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio-Technology 1:784–791

    Article  CAS  Google Scholar 

  • Stowers CC, Cox BM, Rodriguez BA (2014) Development of an industrializable fermentation process for propionic acid production. J Ind Microbiol Biotechnol 41:837–852

    Article  CAS  PubMed  Google Scholar 

  • Sumantran VN, Schweizer HP, Datta P (1990) A novel membrane-associated threonine permease encoded by the tdcC gene of Escherichia coli. J Bacteriol 172:4288–4294

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suwannakham S, Yang ST (2005) Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor. Biotechnol Bioeng 91:325–337

    Article  CAS  PubMed  Google Scholar 

  • Tufvesson P, Ekman A, Sardari RRR, Engdahl K, Tufvesson L (2013) Economic and environmental assessment of propionic acid production by fermentation using different renewable raw materials. Bioresour Technol 149:556–564

    Article  CAS  PubMed  Google Scholar 

  • Vidra A, Nemeth A (2018) Bio-produced propionic acid: a review. Periodica Polytechnica-Chemical Engineering 62:57–67

    Article  CAS  Google Scholar 

  • Volodina E, Schürmann M, Lindenkamp N, Steinbüchel A (2014) Characterization of propionate CoA-transferase from Ralstonia eutropha H16. Appl Microbiol Biotechnol 98:3579–3589

    Article  CAS  PubMed  Google Scholar 

  • Werpy T, Holladay J, White J (2004) Top value added chemicals from biomass: I. results of screening for potential candidates from sugars and synthesis gas. Univ Pa Law Rev 154(3):477

    Google Scholar 

  • Yang H, Wang Z, Lin M, Yang ST (2018) Propionic acid production from soy molasses by Propionibacterium acidipropionici: fermentation kinetics and economic analysis. Bioresour Technol 250:1–9

    Article  CAS  PubMed  Google Scholar 

  • Zhang A, Yang ST (2009) Engineering Propionibacterium acidipropionici for enhanced propionic acid tolerance and fermentation. Biotechnol Bioeng 104:766–773

    CAS  PubMed  Google Scholar 

  • Zhang CL, Qi JS, Li YJ, Fan XG, Xu QY, Chen N, Xie XX (2016) Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng 113:2054–2059

    Article  CAS  PubMed  Google Scholar 

  • Zhuang Z, Song F, Zhao H, Li L, Cao J, Eisenstein E, Herzberg O, Dunaway-Mariano D (2008) Divergence of function in the hot dog fold enzyme superfamily: the bacterial thioesterase YciA. Biochemistry 47:2789–2796

    Article  CAS  PubMed  Google Scholar 

  • Zhuge X, Li J, Shin HD, Liu L, Du G, Chen J (2015) Improved propionic acid production with metabolically engineered Propionibacterium jensenii by an oxidoreduction potential-shift control strategy. Bioresour Technol 175:606–612

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This work was supported by the National Key R & D Program of China (2018YFA0901400), National Natural Science Foundation of China (31770122), and Science & Technology Partnership Program, Ministry of Science and Technology of China (KY201701011).

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Authors and Affiliations

  1. CAS Key Laboratory of Microbial Physiological & Metabolic Engineering and State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

    Chao Ma, Qingxuan Mu, Lei Wang, Yanan Shi, Shasha Zhang, Yanfen Xue, Yong Tao, Yanhe Ma & Bo Yu

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Chao Ma, Qingxuan Mu, Lei Wang, Yanan Shi & Shasha Zhang

  3. IBG-1: Biotechnology, Institute of Bio- and Geosciences, Forschungszentrum Jülich, Jülich, Germany

    Lingfeng Zhu

  4. China-Thailand Joint Laboratory on Microbial Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

    Bo Yu

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  1. Chao Ma
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  2. Qingxuan Mu
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  4. Yanan Shi
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  9. Yanhe Ma
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  10. Bo Yu
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Correspondence to Yanhe Ma or Bo Yu.

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This work has been included in patent applications by the Institute of Microbiology, Chinese Academy of Sciences.

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Ma, C., Mu, Q., Wang, L. et al. Bio-production of high-purity propionate by engineering l-threonine degradation pathway in Pseudomonas putida. Appl Microbiol Biotechnol 104, 5303–5313 (2020). https://doi.org/10.1007/s00253-020-10619-7

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  • DOI: https://doi.org/10.1007/s00253-020-10619-7

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