Expression of Alternative Nitrogenases in Rhodopseudomonas palustris Is Enhanced Using an Optimized Genetic Toolset for Rapid, Markerless Modifications

被引:10
作者
du Toit, Jan-Pierre [1 ]
Lea-Smith, David J. [2 ,3 ]
Git, Anna [3 ]
Hervey, John R. D. [3 ]
Howe, Christopher J. [3 ]
Pott, Robert W. M. [1 ]
机构
[1] Stellenbosch Univ, Dept Proc Engn, ZA-7600 Stellenbosch, South Africa
[2] Univ East Anglia, Sch Biol Sci, Norwich NR4 7TJ, Norfolk, England
[3] Univ Cambridge, Dept Biochem, Cambridge CB2 1QW, England
来源
ACS SYNTHETIC BIOLOGY | 2021年 / 10卷 / 09期
基金
新加坡国家研究基金会;
关键词
biohydrogen; Rhodopseudomonas palustris; genetic toolset; alternative nitrogenases; gene overexpression; insertional mutagenesis; POSTTRANSLATIONAL MODIFICATION; HYDROGEN-PRODUCTION; RNA; SYSTEM; LIGHT; PHOTOSYNTHESIS; IDENTIFICATION; CONSTRUCTION; SPHAEROIDES; INCREASES;
D O I
10.1021/acssynbio.0c00496
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The phototrophic bacterium Rhodopseudomonas palustris is emerging as a promising biotechnological chassis organism, due to its resilience to a range of harsh conditions, a wide metabolic repertoire, and the ability to quickly regenerate ATP using light. However, realization of this promise is impeded by a lack of efficient, rapid methods for genetic modification. Here, we present optimized tools for generating chromosomal insertions and deletions employing electroporation as a means of transformation. Generation of markerless strains can be completed in 12 days, approximately half the time for previous conjugation-based methods. This system was used for overexpression of alternative nitrogenase isozymes with the aim of improving biohydrogen productivity. Insertion of the pucBa promoter upstream of vnf and anf nitrogenase operons drove robust overexpression up to 4000-fold higher than wild-type. Transcript quantification was facilitated by an optimized high-quality RNA extraction protocol employing lysis using detergent and heat. Overexpression resulted in increased nitrogenase protein levels, extending to superior hydrogen productivity in bioreactor studies under nongrowing conditions, where promoter-modified strains better utilized the favorable energy state created by reduced competition from cell division. Robust heterologous expression driven by the pucBa promoter is thus attractive for energy-intensive biosyntheses suited to the capabilities of R. palustris. Development of this genetic modification toolset will accelerate the advancement of R. palustris as a biotechnological chassis organism, and insights into the effects of nitrogenase overexpression will guide future efforts in engineering strains for improved hydrogen production.
引用
收藏
页码:2167 / 2178
页数:12
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