Pioneering precision in markerless strain development for Synechococcus sp. PCC 7002.
Synechococcus
Counter selection
Markerless
Phenylalanyl-tRNA synthetase
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
08 Oct 2024
08 Oct 2024
Historique:
received:
12
08
2024
accepted:
27
09
2024
medline:
9
10
2024
pubmed:
9
10
2024
entrez:
8
10
2024
Statut:
epublish
Résumé
Marine cyanobacteria such as Picosynechococcus sp. (formerly called Synechococcus sp.) PCC 7002 are promising chassis for photosynthetic production of commodity chemicals with low environmental burdens. Genetic engineering of cyanobacteria conventionally employs antibiotic resistance markers. However, limited availability of antibiotic-resistant markers is a problem for highly multigenic strain engineering. Although several markerless genetic manipulation methods have been developed for PCC 7002, they often lack versatility due to the requirement of gene disruption in the host strain. To achieve markerless transformation in Synechococcus sp. with no requirements for the host strain, this study developed a method in which temporarily introduces a mutated phenylalanyl-tRNA synthetase gene (pheS) into the genome for counter selection. Amino acid substitutions in the PheS that cause high susceptibility of PCC 7002 to the phenylalanine analog p-chlorophenylalanine were examined, and the combination of T261A and A303G was determined as the most suitable mutation. The mutated PheS-based selection was utilized for the markerless knockout of the nblA gene in PCC 7002. In addition, the genetic construct containing the lldD and lldP genes from Escherichia coli was introduced into the ldhA gene site using the counter selection strategy, resulting in a markerless recombinant strain. The repeatability of this method was demonstrated by the double markerless knockin recombinant strain, suggesting it will be a powerful tool for multigenic strain engineering of cyanobacteria.
Identifiants
pubmed: 39379966
doi: 10.1186/s12934-024-02543-6
pii: 10.1186/s12934-024-02543-6
doi:
Substances chimiques
Phenylalanine-tRNA Ligase
EC 6.1.1.20
Bacterial Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
268Subventions
Organisme : Japan Science and Technology Agency
ID : JPMJAL1608
Organisme : Japan Science and Technology Agency
ID : JPMJAL1608
Organisme : Japan Science and Technology Agency
ID : JPMJAL1608
Informations de copyright
© 2024. The Author(s).
Références
Knoot CJ, Ungerer J, Wangikar PP, Pakrasi HB. Cyanobacteria: promising biocatalysts for sustainable chemical production. J Biol Chem. 2018;293:5044–52.
doi: 10.1074/jbc.R117.815886
pubmed: 28972147
Lin PC, Pakrasi HB. Engineering cyanobacteria for production of terpenoids. Planta. 2019;249:145–54.
doi: 10.1007/s00425-018-3047-y
pubmed: 30465115
Liu XF, Xie H, Roussou S, Lindblad P. Current advances in engineering cyanobacteria and their applications for photosynthetic butanol production. Curr Opin Biotechnol. 2022;73:143–50.
doi: 10.1016/j.copbio.2021.07.014
pubmed: 34411807
Hidese R, Matsuda M, Osanai T, Hasunuma T, Kondo A. Malic enzyme facilitates d-lactate production through increased pyruvate supply during anoxic dark fermentation in Synechocystis sp. PCC 6803. ACS Synth Biol. 2020;9:260–8.
doi: 10.1021/acssynbio.9b00281
pubmed: 32004431
Hasunuma T, Matsuda M, Kato Y, Vavricka CJ, Kondo A. Temperature enhanced succinate production concurrent with increased central metabolism turnover in the cyanobacterium Synechocystis sp. PCC 6803. Metab Eng. 2018;48:109–20.
doi: 10.1016/j.ymben.2018.05.013
pubmed: 29847778
Davies FK, Work VH, Beliaev AS, Posewitz MC. Engineering limonene and bisabolene production in wild type and a glycogen-deficient mutant of Synechococcus sp. PCC 7002. Front Bioeng Biotechnol. 2014;2:21.
doi: 10.3389/fbioe.2014.00021
pubmed: 25152894
pmcid: 4126464
Nomura CT, Sakamoto T, Bryant DA. Roles for heme-copper oxidases in extreme high-light and oxidative stress response in the cyanobacterium Synechococcus sp. PCC 7002. Arch Microbiol. 2006;185:471–9.
doi: 10.1007/s00203-006-0107-7
pubmed: 16775753
Kimura A, Hamada T, Morita EH, Hayashi H. A high temperature-sensitive mutant of Synechococcus sp. PCC 7002 with modifications in the endogenous plasmid, pAQ1. Plant Cell Physiol. 2002;43:217–23.
doi: 10.1093/pcp/pcf022
pubmed: 11867701
Viola S, Ruhle T, Leister D. A single vector-based strategy for marker-less gene replacement in Synechocystis sp. PCC 6803. Microb Cell Fact 2014, 13.
Ungerer J, Pakrasi HB. Cpf1 is a versatile tool for CRISPR genome editing across diverse species of Cyanobacteria. Sci Rep 2016, 6.
Wendt KE, Ungerer J, Cobb RE, Zhao H, Pakrasi HB. CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. Microb Cell Fact. 2016;15:1–8.
doi: 10.1186/s12934-016-0514-7
Baldanta S, Guevara G, Navarro-Llorens JM. SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria. Microb Cell Fact 2022, 21.
Niu TC, Lin GM, Xie LR, Wang ZQ, Xing WY, Zhang JY, Zhang CC. Expanding the potential of CRISPR-Cpf1-based genome editing technology in the cyanobacterium Anabaena PCC 7120. ACS Synth Biol. 2019;8:170–80.
doi: 10.1021/acssynbio.8b00437
pubmed: 30525474
Begemann MB, Zess EK, Walters EM, Schmitt EF, Markley AL, Pfleger BF. An organic acid based counter selection system for Cyanobacteria. PLoS ONE 2013, 8.
Kojima K, Keta S, Uesaka K, Kato A, Takatani N, Ihara K, Omata T, Aichi M. A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase. Appl Microbiol Biotechnol. 2016;100:10107–13.
doi: 10.1007/s00253-016-7850-8
pubmed: 27704180
pmcid: 5102962
Carr JF, Danziger ME, Huang AL, Dahlberg AE, Gregory ST. Engineering the genome of Thermus thermophilus using a counterselectable marker. J Bacteriol. 2015;197:1135–44.
doi: 10.1128/JB.02384-14
pubmed: 25605305
pmcid: 4336342
Wang YC, Yuan LS, Tao HX, Jiang W, Liu CJ. pheS* as a counter-selectable marker for marker-free genetic manipulations in Bacillus anthracis. J Microbiol Methods. 2018;151:35–8.
doi: 10.1016/j.mimet.2018.05.024
pubmed: 29859216
Zhou CY, Shi LL, Ye B, Feng HC, Zhang J, Zhang RF, Yan X. pheS*, an effective host-genotype-independent counter-selectable marker for marker-free chromosome deletion in Bacillus amyloliquefaciens. Appl Microbiol Biotechnol. 2017;101:217–27.
doi: 10.1007/s00253-016-7906-9
pubmed: 27730334
Xin YP, Guo TT, Mu YL, Kong J. Development of a counterselectable seamless mutagenesis system in lactic acid bacteria. Microb Cell Fact 2017, 16.
Xie ZJ, Okinaga T, Qi FX, Zhang ZJ, Merritt J. Cloning-independent and counterselectable markerless mutagenesis system in Streptococcus mutans. Appl Environ Microbiol. 2011;77:8025–33.
doi: 10.1128/AEM.06362-11
pubmed: 21948849
pmcid: 3208986
Zhang S, Zou ZZ, Kreth J, Merritt J. Recombineering in Streptococcus mutans using direct repeat-mediated cloning-independent markerless mutagenesis (DR-CIMM). Front Cell Infect Microbiol 2017, 7.
Gao GJ, Wei D, Li G, Chen P, Wu LJ, Liu SG, Zhang YL. Highly effective markerless genetic manipulation of Streptococcus suis using a mutated PheS-based counterselectable marker. Front Microbiol 2022, 13.
Barrett AR, Kang Y, Inamasu KS, Son MS, Vukovich JM, Hoang TT. Genetic tools for allelic replacement in Burkholderia species. Appl Environ Microbiol. 2008;74:4498–508.
doi: 10.1128/AEM.00531-08
pubmed: 18502918
pmcid: 2493169
Kast P, Hennecke H. Amino acid substrate specificity of Escherichia coli phenylalanyl-tRNA synthetase alterd by distinct mutations. J Mol Biol. 1991;222:99–124.
doi: 10.1016/0022-2836(91)90740-W
pubmed: 1942071
Miyazaki K. Molecular engineering of a PheS counterselection marker for improved operating efficiency in Escherichia coli. Biotechniques. 2015;58:86–8.
doi: 10.2144/000114257
pubmed: 25652032
Kato Y, Inabe K, Haraguchi Y, Shimizu T, Kondo A, Hasunuma T. l-Lactate treatment by photosynthetic cyanobacteria expressing heterogeneous l-lactate dehydrogenase. Sci Rep. 2023;13:7249.
doi: 10.1038/s41598-023-34289-3
pubmed: 37142758
pmcid: 10160077
Collier JL, Grossman AR. A small polypeptide triggers complete degradation of light-harvesting phycobiliproteins in nutrient-deprived Cyanobacteria. EMBO J. 1994;13:1039–47.
doi: 10.1002/j.1460-2075.1994.tb06352.x
pubmed: 8131738
pmcid: 394911
Li H, Sherman LA. Characterization of Synechocystis sp. strain PCC 6803 and ∆nbl mutants under nitrogen-deficient conditions. Arch Microbiol. 2002;178:256–66.
doi: 10.1007/s00203-002-0446-y
pubmed: 12209258
Yu JJ, Liberton M, Cliften PF, Head RD, Jacobs JM, Smith RD, Koppenaal DW, Brand JJ, Pakrasi HB. Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO
Takatani N, Use K, Kato A, Ikeda K, Kojima K, Aichi M, Maeda S, Omata T. Essential role of acyl-ACP synthetase in acclimation of the cyanobacterium Synechococcus elongatus Strain PCC 7942 to high-light conditions. Plant Cell Physiol. 2015;56:1608–15.
doi: 10.1093/pcp/pcv086
pubmed: 26063393
Schuster CF, Howard SA, Grundling A. Use of the counter selectable marker PheS* for genome engineering in Staphylococcus aureus. Microbiology-Sgm. 2019;165:572–84.
doi: 10.1099/mic.0.000791
Mermershtain I, Finarov I, Klipcan L, Kessler N, Rozenberg H, Safro MG. Idiosyncrasy and identity in the prokaryotic phe-system: Crystal structure of E. coli phenylalanyl-tRNA synthetase complexed with phenylalanine and AMP. Protein Sci. 2011;20:160–7.
doi: 10.1002/pro.549
pubmed: 21082706