Abstract
With the reduction in oil reserves and steady increases in the price of oil, alternative carbon sources like methanol are promising, but an efficient conversion process to fuels and other chemicals is still desired. In this study, we demonstrated for the first time the production of lactic acid from methanol using a lactate dehydrogenase copy number amplifying strategy in Pichia pastoris. We engineered methylotrophic yeast (Pichia pastoris) producing d-lactic acid by d-lactate dehydrogenase gene (d-LDH) integration into the non-transcribed spacer of the ribosomal DNA (rDNA) locus and post-transformational amplification. The resultant engineered strains GS115/S8/Z3 and GS115/S16/Z3 produced 3.48 and 3.26 g/L of d-lactic acid from methanol, respectively, in a 96-h test tube fermentation. To our knowledge, this is the first report about d-lactic acid production from methanol by an engineered P. pastoris strain. The technique of gene integration into the rDNA locus and post-transformational gene amplification could be useful for metabolic engineering in P. pastoris, and the chemical production from methanol by engineered P. pastoris represents a promising industrial technology.
Graphical abstract
Similar content being viewed by others
References
Avigad G (1983) A simple spectrophotometric determination of formaldehyde and other aldehydes: application to periodate-oxidized glycol systems. Anal Biochem 134:499–504
Baek SH, Kwon EY, Kim YH, Hahn JS (2016) Metabolic engineering and adaptive evolution for efficient production of D-lactic acid in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 100:2737–2748
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Casal M, Paiva S, Andrade RP, Gancedo C, Leao C (1999) The lactate-proton symport of Saccharomyces cerevisiae is encoded by JEN1. J Bacteriol 181:2620–2623
Celik E, Calik P, Oliver SG (2010) Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: effects of methanol feeding rate. Biotechnol Bioeng 105:317–329
Cregg JM, Barringer KJ, Hessler AY, Madden KR (1985) Pichia pastoris as a host system for transformations. Mol Cell Biol 5:3376–3385
de Lima PB, Mulder KC, Melo NT, Carvalho LS, Menino GS, Mulinari E, de Castro VH, Dos Reis TF, Goldman GH, Magalhaes BS, Parachin NS (2016) Novel homologous lactate transporter improves l-lactic acid production from glycerol in recombinant strains of Pichia pastoris. Microb Cell Fact 15:158
De Bhowmick G, Sarmah AK, Sen R (2018) Lignocellulosic biorefinery as a model for sustainable development of biofuels and value added products. Bioresour Technol 247:1144–1154
Ganley AR, Ide S, Saka K, Kobayashi T (2009) The effect of replication initiation on gene amplification in the rDNA and its relationship to aging. Mol Cell 35:683–693
Gao C, Ma C, Xu P (2011) Biotechnological routes based on lactic acid production from biomass. Biotechnol Adv 29:930–939
Garvie EI (1980) Bacterial lactate dehydrogenases. Microbiol Rev 44:106–139
Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741–750
Gunji Y, Yasueda H (2006) Enhancement of l-lysine production in methylotroph Methylophilus methylotrophus by introducing a mutant LysE exporter. J Biotechnol 127:1–13
Hofvendahl K, Hahn–Hägerdal B (2000) Factors affecting the fermentative lactic acid production from renewable resources1. Enzyme Microb Technol 26:87–107
Ishida N, Suzuki T, Tokuhiro K, Nagamori E, Onishi T, Saitoh S, Kitamoto K, Takahashi H (2006) d-Lactic acid production by metabolically engineered Saccharomyces cerevisiae. J Biosci Bioeng 101:172–177
Kawaguchi H, Hasunuma T, Ogino C, Kondo A (2016) Bioprocessing of bio-based chemicals produced from lignocellulosic feedstocks. Curr Opin Biotechnol 42:30–39
Kim SW, Kim P, Lee HS, Kim JH (1996) High production of Poly-β-hydroxybutyrate (PHB) from Methylobacterium organophilum under potassium limitation. Biotechnol Lett 18:25–30
Klabunde J, Diesel A, Waschk D, Gellissen G, Hollenberg CP, Suckow M (2002) Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha. Appl Microbiol Biotechnol 58:797–805
Laopaiboon P, Thani A, Leelavatcharamas V, Laopaiboon L (2010) Acid hydrolysis of sugarcane bagasse for lactic acid production. Bioresour Technol 101:1036–1043
Lopes TS, Klootwijk J, Veenstra AE, van der Aar PC, van Heerikhuizen H, Raué HA, Planta RJ (1989) High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene 79:199–206
Lopes TS, Hakkaart GJ, Koerts BL, Raue HA, Planta RJ (1991) Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae. Gene 105:83–90
Marx H, Mecklenbrauker A, Gasser B, Sauer M, Mattanovich D (2009) Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus. FEMS Yeast Res 9:1260–1270
Moon HY, Lee DW, Sim GH, Kim HJ, Hwang JY, Kwon MG, Kang BK, Kim JM, Kang HA (2016) A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts. J Biotechnol 233:190–199
Motoyama H, Anazawa H, Katsumata R, Araki K, Teshiba S (1993) Amino acid production from methanol by Methylobacillus glycogenes mutants: Isolation of l-glutamic acid hyper-producing mutants from M. glycogenes strains, and derivation of l-threonine and l-lysine-producing mutants from them. Biosci Biotechnol Biochem 57:82–87
Pacheco A, Talaia G, Sa-Pessoa J, Bessa D, Goncalves MJ, Moreira R, Paiva S, Casal M, Queiros O (2012) Lactic acid production in Saccharomyces cerevisiae is modulated by expression of the monocarboxylate transporters Jen1 and Ady2. FEMS Yeast Res 12:375–381
Paiva S, Devaux F, Barbosa S, Jacq C, Casal M (2004) Ady2p is essential for the acetate permease activity in the yeast Saccharomyces cerevisiae. Yeast 21:201–210
Pfeifenschneider J, Brautaset T, Wendisch VF (2017) Methanol as carbon substrate in the bio-economy: metabolic engineering of aerobic methylotrophic bacteria for production of value-added chemicals. Biofuel Bioprod Biorefin 11:719–731
Sauer M, Porro D, Mattanovich D, Branduardi P (2008) Microbial production of organic acids: expanding the markets. Trends Biotechnol 26:100–108
Schrader J, Schilling M, Holtmann D, Sell D, Filho MV, Marx A, Vorholt JA (2009) Methanol-based industrial biotechnology: current status and future perspectives of methylotrophic bacteria. Trends Biotechnol 27:107–115
Schroer K, Peter Luef K, Stefan Hartner F, Glieder A, Pscheidt B (2010) Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation. Metab Eng 12:8–17
Schwarzhans JP, Luttermann T, Geier M, Kalinowski J, Friehs K (2017) Towards systems metabolic engineering in Pichia pastoris. Biotechnol Adv 35:681–710
Sirirote P, Tsuneo Y, Shoichi S (1988) l-Serine production from methanol and glycine with an immobilized methylotroph. J Ferment Technol 66:291–297
Steinborn G, Böer E, Scholz A, Tag K, Kunze G, Gellissen G (2006) Application of a wide-range yeast vector (CoMed™) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts. Microb Cell Fact 5:33
Tokuhiro K, Ishida N, Nagamori E, Saitoh S, Onishi T, Kondo A, Takahashi H (2009) Double mutation of the PDC1 and ADH1 genes improves lactate production in the yeast Saccharomyces cerevisiae expressing the bovine lactate dehydrogenase gene. Appl Microbiol Biotechnol 82:883–890
Tsuji H (2005) Poly(lactide) stereocomplexes: formation, structure, properties, degradation, and applications. Macromol Biosci 5:569–597
Wendisch VF, Brito LF, Gil Lopez M, Hennig G, Pfeifenschneider J, Sgobba E, Veldmann KH (2016) The flexible feedstock concept in industrial biotechnology: Metabolic engineering of Escherichia coli, Corynebacterium glutamicum, Pseudomonas, Bacillus and yeast strains for access to alternative carbon sources. J Biotechnol 234:139–157
Wu S, Letchworth GJ (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36:152–154
Yamada R, Tanaka T, Ogino C, Kondo A (2010) Gene copy number and polyploidy on products formation in yeast. Appl Microbiol Biotechnol 88:849–857
Yamada R, Hasunuma T, Kondo A (2013) Endowing non-cellulolytic microorganisms with cellulolytic activity aiming for consolidated bioprocessing. Biotechnol Adv 31:754–763
Yamada R, Kimoto Y, Ogino H (2016) Combinatorial library strategy for strong overexpression of the lipase from Geobacillus thermocatenulatus on the cell surface of yeast Pichia pastoris. Biochem Eng J 113:7–11
Yamada R, Wakita K, Mitsui R, Ogino H (2017a) Enhanced d-lactic acid production by recombinant Saccharomyces cerevisiae following optimization of the global metabolic pathway. Biotechnol Bioeng 114:2075–2084
Yamada R, Wakita K, Ogino H (2017b) Global metabolic engineering of glycolytic pathway via multicopy integration in Saccharomyces cerevisiae. ACS Synth Biol 6:659–666
Yurimoto H, Oku M, Sakai Y (2011) Yeast methylotrophy: metabolism, gene regulation and peroxisome homeostasis. Int J Microbiol 2011:101298
Acknowledgements
This work was partly supported by Japan Society for the Promotion of Science KAKENHI (Grant Number 18K14069) to RY.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Yamada, R., Ogura, K., Kimoto, Y. et al. Toward the construction of a technology platform for chemicals production from methanol: d-lactic acid production from methanol by an engineered yeast Pichia pastoris. World J Microbiol Biotechnol 35, 37 (2019). https://doi.org/10.1007/s11274-019-2610-4
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-019-2610-4