Isolation of a Rhodobacter sphaeroides mutant with enhanced hydrogen production capacity from transposon mutagenesis by NH4+ nitrogen resource

https://doi.org/10.1016/j.ijhydene.2018.01.179Get rights and content

Highlights

  • The transposon was employed for NH4+ resistant controlgene from CBB deletion firstly.

  • Disruption of trmB could partially derepress the ammonium inhibition of nitrogenase.

  • The H2 production rate and nitrogenase activity of mutant (trmB) were enhanced.

Abstract

In order to mitigate the ammonia-repression effect of nitrogenase in photosynthetic bacteria, we screened a library of transposon insertion mutants that showed better hydrogen production performance under ammonia-repressed conditions, revealing a relevant regulatory gene. The transposon plasmid pRL27 was engaged for transposon mutagenesis in a cbbR nullmutant of photosynthetic bacteria that has poor growth in the presence of ammonia, and mutants with better growth characteristics were successfully isolated. The hydrogen production yield and rate of one mutant (WRR04) were improved by 14.16% and 31.38%, respectively. BLAST analysis revealed that the interrupted DNA sequence encodes trmB, a transcriptional regulator. Additionally, the mutants WTF05 (trmB-) and WTR06 (trmB-, cbbR-) were constructed, and their H2 production rates were increased remarkably by 54.72% and 41.44% comparing with the wild strain, respectively. The enhancement of nitrogenase activity demonstrates that the rupture of trmB could partly de-repress the activity of nitrogenase inhibited by ammonium for Rhodobacter sphaeroides.

Introduction

The highest hydrogen production output and substrate conversion efficiency reported in Rhodococcus spp. was obtained by dark-photo combining fermentation [1], [2]. Nevertheless, the low rate of H2 output in fermentation by photoheterotrophic bacteria was the bottleneck of this two-stage H2 production procedure [3]. Ammonium inhibition of nitrogenase activity is one of the reasons for low hydrogen production rates, since ammonium is commonly present in the dark fermentation culture broth [4]. The NH4+ is a pronounced inhibitor of nitrogenase, and hence an inhibitor of H2 production by anaerobic photo synthetic bacteria [5]. So as to remove ammonium inhibition of nitrogenase, many enzymes related to ammonium regulation have been investigated. Zhihua Zhou and coworkers found that knocking out the glnA gene, which encodes glutamine synthetase, can de-repress the expression of nifA and nitrogenase genes. They suggested that glutamine synthetase (GS) is essential for ammonium repression, and the deletion of glnA1 is a workable approach to relieve the inhibition of nitrogenase gene expression in Rhodobacter sphaeroides [6]. Some studies found that mutations in some amino acids in nifA can increase the tolerance of nifA to ammonia nitrogen and thus de-repress the expression of nitrogenase [7], [8], [9]. In our previous study, we found that the rupture of spbA in a wild strain of Rhodobacter sphaeroides could partly de-repress NH4+ suppression to the activity of nitrogenase, which indicates that spbA has some certain negative correlation effects on the expression of nitrogenase with a certain concentration of ammonium [4]. However, the regulatory mechanism of hydrogen production by nitrogenase involves a very complex network, and the genes that can de-repress the ammonium effect on nitrogenase need further exploration.

In this study, the transposon plasmid pRL27 [10] was used to obtain insertion-disrupted mutants which could grow well when NH4+ and succinate were used as the nitrogen and carbon source under light anoxygenic conditions. A mutant strain with an interruption of trmB was discovered to be able to maintain hydrogen production under high-NH4+ conditions (Nitrogen sources composition: 0.12 mM ammonium and 0.48 mM glutamic acid). At present, the operational roles of the TrmB family members by the observational grounds are limited in Thermococcales [11], halophilic archaeon H. salinarum and crenarchaeon S. acidocaldarius [12]. TrmB family members were well characterized as global transcriptional regulators for sugar transporters and various metabolic pathways, and chromosomal stabilizer in archaeal and bacterial genomes [13]. Hence, investigations of the TrmB family members into the characterization of other bacterial need to expand to different type in prokaryotes. To our best knowledge, this is the first study to report a new function of TrmB in terms of mitigating ammonium inhibition.

Section snippets

Inoculum and bacteria

The expression plasmids and bacteria constructed and used in this paper were listed in Table 1. Medium Luria-Bertani (LB) was used to culture the Escherichia coli strains as described previously [14]. Antibiotic selection of E. coli strains carrying recombinant plasmids was performed by growing the bacteria at 37 °C aerobically on LB plates comprising 2% agar powder, supplemented with antibiotics as described previously [15]. Modified Sistrom's succinate minimal (MedA) medium and MPYE were used

Screening for ammonium-resistant mutants

When R. sphaeroides grows photoheterotrophically, excess reducing equivalents are produced, which needs to be dissipated in order to maintain a balanced redox potential. CO2 fixation or hydrogen production can reuse the excess reducing equivalents served as the electron sink out of the light-mediated exogenous substrates oxidation [2], [25]. Thus, CO2 fixation or hydrogen production will occur to reuse the excess reducing equivalents and thus make cell grow photoheterotrophically [15]. CO2 is

Conclusions

The mutant WRR04 obtained by pRL27 transposon mutagenesis in a high-throughput screen showed better H2 generation performance in the presence of ammonia. The trmB gene in WR02 (cbbR) was shown to be disrupted by transposon insertion via sequencing and alignment. The function of trmB was verified by suicide-plasmid knockout in the wild-type. The H2 production yield and its evolution rate of the trmB mutant were both improved, improving that the nitrogenase inhibition effect was alleviated to

Acknowledgements

This study was funded by the Shaanxi Provincial Science and Technology Research Projects (No.2017JQ5057 and No.2011K08-05), the Scientific Research Plan Project of Shaanxi Education Department (No.17JK0098), the Science and Technology Planning Project of Sichuan ProvinceNo. 2016JZ0018)and the Major Science and Technology Special Program in Xianyang (No.2016k01-15).

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