Transcription regulation strategies in methylotrophs: progress and challenges

Abstract

As a promising industrial microorganism, methylotroph is capable of using methane or methanol as the sole carbon source natively, which has been utilized in the biosynthesis of various bioproducts. However, the relatively low efficiency of carbon conversion has become a limiting factor throughout the development of methanotrophic cell factories due to the unclear genetic background. To better highlight their advantages in methane or methanol-based biomanufacturing, some metabolic engineering strategies, including upstream transcription regulation projects, are being popularized in methylotrophs. In this review, several strategies of transcription regulations applied in methylotrophs are summarized and their applications are discussed and prospected.

Keywords

Methylotrophs / C1 bioconversion / Transcription regulation / Transcription factor / Promoter / CRISPR

References

[1] Amann E, Ochs B, Abel K-J. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene, 1988, 69(2): 301-315.

[2] Ameruoso A, Villegas Kcam MC, Cohen KP, Chappell J. Activating natural product synthesis using CRISPR interference and activation systems in Streptomyces. Nucleic Acids Res, 2022, 50(13): 7751-7760.

[3] Amuel C, Gellissen G, Hollenberg CP, Suckow M. Analysis of heat shock promoters in Hansenula polymorpha: The TPS1 promoter, a novel element for heterologous gene expression. Biotechnol Bioprocess Eng, 2000, 5(4): 247-252.

[4] Baghban R, Farajnia S, Rajabibazl M, Ghasemi Y, Mafi A, Hoseinpoor R, Rahbarnia L, Aria M. Yeast expression systems: overview and recent advances. Mol Biotechnol, 2019, 61(5): 365-384.

[5] Blazeck J, Alper HS. Promoter engineering: recent advances in controlling transcription at the most fundamental level. Biotechnol J, 2013, 8(1): 46-58.

[6] Blazeck J, Garg R, Reed B, Alper HS. Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters. Biotechnol Bioeng, 2012, 109(11): 2884-2895.

[7] Cai P, Duan X, Wu X, Gao L, Ye M, Zhou YJ. Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris. Nucleic Acids Res, 2021, 49(13): 7791-7805.

[8] Carere CR, McDonald B, Peach HA, Greening C, Gapes DJ, Collet C, Stott MB. Hydrogen oxidation influences glycogen accumulation in a verrucomicrobial methanotroph. Front Microbiol, 2019, 10: 1873.

[9] Cazier AP, Blazeck J. Advances in promoter engineering: novel applications and predefined transcriptional control. Biotechnol J, 2021, 16(10

[10] Cheng T, Wang L, Sun C, Xie C. Optimizing the downstream MVA pathway using a combination optimization strategy to increase lycopene yield in Escherichia coli. Microb Cell Fact, 2022

[11] Chistoserdova L, Kalyuzhnaya MG, Lidstrom ME. The expanding world of methylotrophic metabolism. Annu Rev Microbiol, 2009, 63: 477-499.

[12] Cho S, Lee YS, Chai H, Lim SE, Na JG, Lee J. Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z. Biotechnol Biofuels Bioprod, 2022, 15(1): 5.

[13] Cui L, Vigouroux A, Rousset F, Varet H, Khanna V, Bikard D. A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nat Commun, 2018, 9(1): 1912.

[14] Czarnek M, Bereta J. The CRISPR-Cas system—from bacterial immunity to genome engineering. Postepy Hig Med Dosw (online), 2016, 70: 901-916.

[15] Dalvie NC, Leal J, Whittaker CA, Yang Y, Brady JR, Love KR, Love JC. Host-informed expression of CRISPR guide RNA for genomic engineering in Komagataella phaffii. ACS Synth Biol, 2020, 9(1): 26-35.

[16] Dam B, Dam S, Blom J, Liesack W. Genome analysis coupled with physiological studies reveals a diverse nitrogen metabolism in Methylocystis sp. strain SC2. PLoS ONE, 2013, 8(10): e74767.

[17] de Boer HA, Comstock LJ, Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci USA, 1983, 80(1): 21-25.

[18] Deng C, Lv X, Li J, Zhang H, Liu Y, Du G, Amaro RL, Liu L. Synergistic improvement of N-acetylglucosamine production by engineering transcription factors and balancing redox cofactors. Metab Eng, 2021, 67: 330-346.

[19] Deng C, Wu Y, Lv X, Li J, Liu Y, Du G, Chen J, Liu L. Refactoring transcription factors for metabolic engineering. Biotechnol Adv, 2022, 57.

[20] Du X, Li Y, Xia YL, Ai SM, Liang J, Sang P, Ji XL, Liu SQ (2016) Insights into protein-ligand interactions: mechanisms, models, and methods. Int J Mol Sci 17(2)

[21] Duan X, Gao J, Zhou YJ. Advances in engineering methylotrophic yeast for biosynthesis of valuable chemicals from methanol. Chinese Chem Lett, 2018, 29(5): 681-686.

[22] Erden-Karaoglan F, Karaoglan M, Yilmaz G, Yilmaz S, Inan M. Deletion analysis of Pichia pastoris alcohol dehydrogenase 2 (ADH2) promoter and development of synthetic promoters. Biotechnol J, 2022, 17(2

[23] Ergun BG, Berrios J, Binay B, Fickers P (2021) Recombinant protein production in Pichia pastoris: from transcriptionally redesigned strains to bioprocess optimization and metabolic modelling. FEMS Yeast Res. 21(7)

[24] Fabarius JT, Wegat V, Roth A, Sieber V. Synthetic methylotrophy in yeasts: towards a circular bioeconomy. Trends Biotechnol, 2021, 39(4): 348-358.

[25] Fisher AL, Ohsako S, Caudy M. The WRPW motif of the hairy-related basic helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and protein-protein interaction domain. Mol Cell Biol, 1996, 16(6): 2670-2677.

[26] Garg S, Clomburg JM, Gonzalez R. A modular approach for high-flux lactic acid production from methane in an industrial medium using engineered Methylomicrobium buryatense 5GB1. J Ind Microbiol Biotechnol, 2018, 45(6): 379-391.

[27] Garg S, Wu H, Clomburg JM, Bennett GN. Bioconversion of methane to C-4 carboxylic acids using carbon flux through acetyl-CoA in engineered Methylomicrobium buryatense 5GB1C. Metab Eng, 2018, 48: 175-183.

[28] Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell, 2013, 154(2): 442-451.

[29] Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M, Weissman JS. Genome-scale CRISPR-Mediated control of gene repression and activation. Cell, 2014, 159(3): 647-661.

[30] Guo S, Zhang T, Chen Y, Yang S, Fei Q. Transcriptomic profiling of nitrogen fixation and the role of NifA in Methylomicrobium buryatense 5GB1. Appl Microbiol Biotechnol, 2022, 106(8): 3191-3199.

[31] Henard CA, Smith H, Dowe N, Kalyuzhnaya MG, Pienkos PT, Guarnieri MT. Bioconversion of methane to lactate by an obligate methanotrophic bacterium. Sci Rep, 2016, 6: 21585.

[32] Hu L, Yang Y, Yan X, Zhang T, Xiang J, Gao Z, Chen Y, Yang S, Fei Q. Molecular mechanism associated with the impact of methane/oxygen gas supply ratios on cell growth of Methylomicrobium buryatense 5GB1 through RNA-Seq. Front Bioeng Biotechnol, 2020, 8: 263.

[33] Hu L, Guo S, Wang B, Fu R, Fan D, Jiang M, Fei Q, Gonzalez R. Bio-valorization of C1 gaseous substrates into bioalcohols: potentials and challenges in reducing carbon emissions. Biotechnol Adv, 2022, 59.

[34] Ishikawa M, Tanaka Y, Suzuki R, Kimura K, Tanaka K, Kamiya K, Ito H, Kato S, Kamachi T, Hori K, Nakanishi S. Real-time monitoring of intracellular redox changes in Methylococcus capsulatus (Bath) for efficient bioconversion of methane to methanol. Bioresour Technol, 2017, 241: 1157-1161.

[35] Ishikawa K, Soejima S, Masuda F, Saitoh S (2021) Implementation of dCas9-mediated CRISPRi in the fission yeast Schizosaccharomyces pombe. G3 (Bethesda) 11(4)

[36] Jia LJ, Zhang KS, Tang K, Meng JY, Zheng C, Feng FY. Methylobacteriumcrusticola sp. nov., isolated from biological soil crusts. Int J Syst Evol Microbiol, 2020, 70(3): 2089-2095.

[37] Killham KEN, Prosser JI. Paul EA. 5—The Prokaryotes. Soil microbiology, ecology and biochemistry, 2007, San Diego: Academic Press, 119-144.

[38] Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature, 2015, 517(7536): 583-588.

[39] Kruse T, Ratnadevi CM, Erikstad HA, Birkeland NK. Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph “Candidatus Methylacidiphilum kamchatkense “strain Kam1 and comparison with its closest relatives. BMC Genomics, 2019, 20(1): 642.

[40] Lee JK, Kim S, Kim W, Kim S, Cha S, Moon H, Hur DH, Kim SY, Na JG, Lee JW, Lee EY, Hahn JS. Efficient production of d-lactate from methane in a lactate-tolerant strain of Methylomonas sp. DH-1 generated by adaptive laboratory evolution. Biotechnol Biofuels, 2019, 12: 234.

[41] Lee HM, Ren J, Yu MS, Kim H, Kim WY, Shen J, Yoo SM, Eyun SI, Na D. Construction of a tunable promoter library to optimize gene expression in Methylomonas sp. DH-1, a methanotroph, and its application to cadaverine production. Biotechnol Biofuels, 2021, 14(1): 228.

[42] Li H, Pham NN, Shen CR, Chang CW, Tu Y, Chang YH, Tu J, Nguyen MTT, Hu YC. Combinatorial CRISPR interference library for enhancing 2,3-BDO production and elucidating key genes in cyanobacteria. Front Bioeng Biotechnol, 2022, 10.

[43] Liang S, Zou C, Lin Y, Zhang X, Ye Y. Identification and characterization of PGCW14: a novel, strong constitutive promoter of Pichia pastoris. Biotechnol Lett, 2013, 35(11): 1865-1871.

[44] Liu Z, Cao L, Fu X, Liang Q, Sun H, Mou H. A multi-functional genetic manipulation system and its use in high-level expression of a beta-mannanase mutant with high specific activity in Pichia pastoris. Microb Biotechnol, 2021, 14(4): 1525-1538.

[45] Madhaiyan M, Poonguzhali S, Lee JS, Saravanan VS, Lee KC, Santhanakrishnan P. Enterobacter arachidis sp. nov., a plant-growth-promoting diazotrophic bacterium isolated from rhizosphere soil of groundnut. Int J Syst Evol Microbiol, 2010, 60(7): 1559-1564.

[46] Manghwar H, Li B, Ding X, Hussain A, Lindsey K, Zhang X, Jin S. CRISPR/Cas systems in genome editing: methodologies and tools for sgRNA design, off-target evaluation, and strategies to mitigate off-target effects. Adv Sci (weinh), 2020, 7(6): 1902312.

[47] Miele R, Barra D, Bonaccorsi di Patti MC. A GATA-type transcription factor regulates expression of the high-affinity iron uptake system in the methylotrophic yeast Pichia pastoris. Arch Biochem Biophys, 2007, 465(1): 172-179.

[48] Mo XH, Zhang H, Wang TM, Zhang C, Zhang C, Xing XH, Yang S. Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis. Appl Microbiol Biotechnol, 2020, 104(10): 4515-4532.

[49] Mustakhimov II, Reshetnikov AS, Glukhov AS, Khmelenina VN, Kalyuzhnaya MG, Trotsenko YA. Identification and characterization of EctR1, a new transcriptional regulator of the ectoine biosynthesis genes in the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. J Bacteriol, 2010, 192(2): 410-417.

[50] Nevoigt E, Kohnke J, Fischer CR, Alper H, Stahl U, Stephanopoulos G. Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol, 2006, 72(8): 5266-5273.

[51] Nguyen AD, Hwang IY, Lee OK, Kim D, Kalyuzhnaya MG, Mariyana R, Hadiyati S, Kim MS, Lee EY. Systematic metabolic engineering of Methylomicrobium alcaliphilum 20Z for 2,3-butanediol production from methane. Metab Eng, 2018, 47: 323-333.

[52] Nguyen AD, Kim D, Lee EY. A comparative transcriptome analysis of the novel obligate methanotroph Methylomonas sp. DH-1 reveals key differences in transcriptional responses in C1 and secondary metabolite pathways during growth on methane and methanol. BMC Genomics, 2019, 20(1): 130.

[53] Nguyen TT, Lee OK, Naizabekov S, Lee EY. Bioconversion of methane to cadaverine and lysine using an engineered type II methanotroph, Methylosinus trichosporium OB3b. Green Chem, 2020, 22(22): 7803-7811.

[54] Nielsen J, Keasling JD. Engineering cellular metabolism. Cell, 2016, 164(6): 1185-1197.

[55] Paget MS. Bacterial sigma factors and anti-sigma factors: structure, function and distribution. Biomolecules, 2015, 5(3): 1245-1265.

[56] Pan X, Tang M, You J, Osire T, Sun C, Fu W, Yi G, Yang T, Yang ST, Rao Z. PsrA is a novel regulator contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in Serratia marcescens. Nucleic Acids Res, 2022, 50(1): 127-148.

[57] Park JN, Sohn MJ, Oh DB, Kwon O, Rhee SK, Hur CG, Lee SY, Gellissen G, Kang HA. Identification of the cadmium-inducible Hansenula polymorpha SEO1 gene promoter by transcriptome analysis and its application to whole-cell heavy-metal detection systems. Appl Environ Microbiol, 2007, 73(19): 5990-6000.

[58] Peters JM, Colavin A, Shi H, Czarny TL, Larson MH, Wong S, Hawkins JS, Lu CHS, Koo BM, Marta E, Shiver AL, Whitehead EH, Weissman JS, Brown ED, Qi LS, Huang KC, Gross CA. A comprehensive, CRISPR-based functional analysis of essential genes in bacteria. Cell, 2016, 165(6): 1493-1506.

[59] Peterson A. CRISPR: express delivery to any DNA address. Oral Dis, 2017, 23(1): 5-11.

[60] Puri AW, Owen S, Chu F, Chavkin T, Beck DA, Kalyuzhnaya MG, Lidstrom ME. Genetic tools for the industrially promising methanotroph Methylomicrobium buryatense. Appl Environ Microbiol, 2015, 81(5): 1775-1781.

[61] Qin X, Qian J, Yao G, Zhuang Y, Zhang S, Chu J. GAP promoter library for fine-tuning of gene expression in Pichia pastoris. Appl Environ Microbiol, 2011, 77(11): 3600-3608.

[62] Schreiber-Agus N, Chin L, Chen K, Torres R, Rao G, Guida P, Skoultchi AI, DePinho RA. An amino-terminal domain of MXI1 mediates anti-myc oncogenic activity and interacts with a homolog of the yeast transcriptional repressor SIN3. Cell, 1995, 80(5): 777-786.

[63] Schultenkamper K, Brito LF, Lopez MG, Brautaset T, Wendisch VF. Establishment and application of CRISPR interference to affect sporulation, hydrogen peroxide detoxification, and mannitol catabolism in the methylotrophic thermophile Bacillus methanolicus. Appl Microbiol Biotechnol, 2019, 103(14): 5879-5889.

[64] Schultenkamper K, Gutle DD, Lopez MG, Keller LB, Zhang L, Einsle O, Jacquot JP, Wendisch VF. Interrogating the role of the two distinct fructose-bisphosphate aldolases of Bacillus methanolicus by site-directed mutagenesis of key amino acids and gene repression by CRISPR interference. Front Microbiol, 2021, 12.

[65] Smirnova AV, Dunfield PF (2018) Differential transcriptional activation of genes encoding soluble methane monooxygenase in a facultative versus an obligate methanotroph. Microorganisms 6(1)

[66] Spitz F, Furlong EE. Transcription factors: from enhancer binding to developmental control. Nat Rev Genet, 2012, 13(9): 613-626.

[67] Stafford GP, Scanlan J, McDonald IR, Murrell JC. rpoN, mmoR and mmoG, genes involved in regulating the expression of soluble methane monooxygenase in Methylosinus trichosporium OB3b. Microbiology (reading), 2003, 149(Pt 7): 1771-1784.

[68] Sun J, Jiang J, Zhai X, Zhu S, Qu Z, Yuan W, Wang Z, Wei C. Coexpression of Kex2 endoproteinase and Hac1 transcription factor to improve the secretory expression of bovine lactoferrin in Pichia pastoris. Biotechnol Bioprocess Eng, 2019, 24(6): 934-941.

[69] Tapscott T, Guarnieri MT, Henard CA (2019) Development of a CRISPR/Cas9 system for Methylococcuscapsulatusinvivo gene. Appl Environ Microbiol 85(11)

[70] Theisen AR, Ali MH, Radajewski S, Dumont MG, Dunfield PF, McDonald IR, Dedysh SN, Miguez CB, Murrell JC. Regulation of methane oxidation in the facultative methanotroph Methylocella silvestris BL2. Mol Microbiol, 2005, 58(3): 682-692.

[71] Vogl T, Kickenweiz T, Pitzer J, Sturmberger L, Weninger A, Biggs BW, Kohler EM, Baumschlager A, Fischer JE, Hyden P, Wagner M, Baumann M, Borth N, Geier M, Ajikumar PK, Glieder A. Engineered bidirectional promoters enable rapid multi-gene co-expression optimization. Nat Commun, 2018, 9(1): 3589.

[72] Waterham HR, Digan ME, Koutz PJ, Lair SV, Cregg JM. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene, 1997, 186(1): 37-44.

[73] Werten MWT, Eggink G, Cohen Stuart MA, de Wolf FA. Production of protein-based polymers in Pichia pastoris. Biotechnol Adv, 2019, 37(5): 642-666.

[74] Wetzel D, Muller JM, Flaschel E, Friehs K, Risse JM. Fed-batch production and secretion of streptavidin by Hansenula polymorpha: evaluation of genetic factors and bioprocess development. J Biotechnol, 2016, 225: 3-9.

[75] Wilson EH, Groom JD, Sarfatis MC, Ford SM, Lidstrom ME, Beck DAC. A computational framework for identifying promoter sequences in nonmodel organisms using RNA-seq data sets. ACS Synth Biol, 2021, 10(6): 1394-1405.

[76] Woolston BM, Emerson DF, Currie DH, Stephanopoulos G. Rediverting carbon flux in Clostridium ljungdahlii using CRISPR interference (CRISPRi). Metab Eng, 2018, 48: 243-253.

[77] Yan X, Chu F, Puri AW, Fu Y, Lidstrom ME. Electroporation-based genetic manipulation in type I methanotrophs. Appl Environ Microbiol, 2016, 82(7): 2062-2069.

[78] Yan C, Yu W, Yao L, Guo X, Zhou YJ, Gao J. Expanding the promoter toolbox for metabolic engineering of methylotrophic yeasts. Appl Microbiol Biotechnol, 2022, 106(9–10): 3449-3464.

[79] Yan C, Yu W, Zhai X, Yao L, Guo X, Gao J, Zhou YJ. Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha. Synth Syst Biotechnol, 2022, 7(1): 498-505.

[80] Yang S, Wang Y, Wei C, Liu Q, Jin X, Du G, Chen J, Kang Z. A new sRNA-mediated posttranscriptional regulation system for Bacillus subtilis. Biotechnol Bioeng, 2018, 115(12): 2986-2995.

[81] Ye RW, Yao H, Stead K, Wang T, Tao L, Cheng Q, Sharpe PL, Suh W, Nagel E, Arcilla D, Dragotta D, Miller ES. Construction of the astaxanthin biosynthetic pathway in a methanotrophic bacterium Methylomonas sp. strain 16a. J Ind Microbiol Biotechnol, 2007, 34(4): 289-299.

[82] Zhan C, Yang Y, Zhang Z, Li X, Liu X, Bai Z (2017) Transcription factor Mxr1 promotes the expression of Aox1 by repressing glycerol transporter 1 in Pichia pastoris. FEMS Yeast Res 17(4)

[83] Zhang F, Wang ZP, Chi Z, Raoufi Z, Abdollahi S, Chi ZM. The changes in TPS1 activity, trehalose content and expression of TPS1 gene in the psychrotolerant yeast Guehomyces pullulans 17–1 grown at different temperatures. Extremophiles, 2013, 17(2): 241-249.

[84] Zhang X, Zhang X, Liang S, Ye Y, Lin Y. Key regulatory elements of a strong constitutive promoter, P_GCW14, from Pichia pastoris. Biotechnol Lett, 2013, 35(12): 2113-2119.

[85] Zhang B, Liu ZQ, Liu C, Zheng YG. Application of CRISPRi in Corynebacterium glutamicum for shikimic acid production. Biotechnol Lett, 2016, 38(12): 2153-2161.

[86] Zhu LP, Song SZ, Yang S. Gene repression using synthetic small regulatory RNA in Methylorubrum extorquens. J Appl Microbiol, 2021, 131(6): 2861-2875.

Funding

National Natural Science Foundation of China(21878241)

Key Research and Development Projects of Shaanxi Province(2021SF-103)

National Key R&D Programs of China(2021YFC2103500)

Science Fund for Distinguished Young Scholars of Shaanxi Province(2022JC-09)