Organizer: Nanjing University of Technology
Co organizer: China Society of Bioengineering
Co organizer: biochemical Professional Committee of China Chemical Society
ISSN：1672-3678 CN：32-1706/Q
Rhodopseudomonas palustris as a microbial cell factory
application
Li Meijie 1, Xia Qingqing 1, Harwood C s1,2, Yang Jianming 1 (1. Photosynthetic carbon sequestration capacity center, School of life sciences, Qingdao Agricultural University; 2. Department of Microbiology, Washington University)
abstract
Rhodopseudomonas palustris is a kind of purple non sulfur bacteria, which has many advantages, such as various metabolic modes, various carbon sources, natural compounds and so on. As a microbial cell factory, Rhodopseudomonas palustris has great research potential and is mainly used in wastewater treatment and aquaculture. In this paper, the author summarizes the research and application of Rhodopseudomonas palustris in the field of microbial cell factories from three aspects, including the diversity of carbon sources, genetic engineering strategies and the application of microbial cell factories, such as in the fields of fuel gas production and terpene compounds, microbial fuel cells, microbial electrosynthesis and Photocatalytic synthesis. At present, Rhodopseudomonas palustris is in the primary stage of development as a microbial cell factory. The author summarizes the difficulties it faces at present, and looks forward to its further research direction. Keywords: Rhodopseudomonas palustris; microbial cell factory; carbon source; hydrogen; methane; terpene compound; electricity producing microorganism
Rhodopseudomonas palustris is a purple non sulfur bacterium (pnsb), belonging to Proteus, oncomelaniaceae and Rhodopseudomonas [1]. Rhodopseudomonas palustris is widely distributed in nature, mainly in the anaerobic water environment with sufficient light, and in lakes, soils, swamps, seas, etc. [2]. Rhodopseudomonas palustris has a variety of metabolic modes, and can grow through four metabolic modes existing in nature, including photosynthetic autotrophy, photosynthetic heterotrophism, chemical autotrophy and chemical heterotrophism (Figure 1) [3].
Fig. 1 four metabolic patterns of Rhodopseudomonas palustris [3]
Fig.1 The four types metabolism of R.palustris[3]
At present, Rhodopseudomonas palustris is mainly used in wastewater treatment and aquaculture (Fig. 2). Rhodopseudomonas palustris is rich in bioactive protein, vitamin, polysaccharide, pantothenic acid, folic acid, etc., which can be used as feed additive [4]; Rhodopseudomonas palustris can improve the oxygen content, stabilize the pH value and purify the aquaculture water environment [5]; Rhodopseudomonas palustris can also improve the immunity of aquatic organisms and prevent diseases. Moreover, Rhodopseudomonas palustris has strong biodegradability, which can degrade many components in animal and plant wastes and industrial wastes, including lignin monomer, nitrogen compounds, chlorides, aromatic compounds, etc. [6]. The whole genome of Rhodopseudomonas palustris was sequenced and a large number of degradation genes were found in its genome [3]. In short, in wastewater treatment and aquaculture industry, Rhodopseudomonas palustris has great research value and is also the focus of current research.
Fig. 2 Application of Rhodopseudomonas palustris
Fig.2 Application of Rhodopseudomonas palustris
In addition to the above applications, in recent years, Rhodopseudomonas palustris has also been applied in other fields, including gas and fuel for hydrogen production (H2) [7], methane production (CH4) [8], terpene compounds such as carotenoids [9], microbial fuel cell (MFC) [10], microbial electrosynthesis [11] and photocatalytic synthesis [12] (Fig. 2).
Rhodopseudomonas palustris shows great potential and application value in the construction of microbial cell factories. In this paper, the author discusses its application as a microbial cell factory in three aspects: carbon diversity; genetic engineering strategy; Rhodopseudomonas palustris as an application field of microbial cell factory. In addition, the current research problems and further research directions are prospected.
1. The diversity of carbon sources is different from other commonly used microbial cell factories. Rhodopseudomonas palustris can utilize carbon sources in waste gas, including small molecular organic acids, alcohols, inorganic salts and aromatic compounds (Table 1). Rhodopseudomonas palustris produces ATP with light as energy source. Therefore, as a microbial cell factory, Rhodopseudomonas palustris has great advantages in energy and carbon sources.
At present, a variety of carbon sources have been reported for the growth of Rhodopseudomonas palustris. First of all, Rhodopseudomonas palustris can decompose and utilize a large number of small molecular organic acids, including acetic acid, butyric acid and lactic acid, which exist in agricultural and industrial wastewater, and produce the target products at the same time of wastewater treatment. Under different organic acids, the growth of Rhodopseudomonas palustris is different, and the ability of producing target products is different. In CH4 producing Rhodopseudomonas palustris, 7 different organic acid salts were added to the culture medium, and when fumaric acid was selected, the CH4 yield was the highest [8]. Different concentrations of carbon source can also affect the yield of target products. In the medium of Rhodopseudomonas palustris producing H2, different concentrations (2-10 mmol / L) of lactic acid were added, and the yield of H2 was different [13].
Secondly, ethanol, crude glycerin, butanol and other alcohols can also be used as carbon sources by Rhodopseudomonas palustris. Crude glycerin is the main by-product of biodiesel production process, so it is generally treated as industrial waste. Based on the consideration of environment and economic benefits, how to use crude glycerin is a breakthrough point in industrial production. The results showed that H 2 could be synthesized from crude glycerol by Rhodopseudomonas palustris.
Thirdly, inorganic salts can be used as carbon source for Rhodopseudomonas palustris. In methanogenic Rhodopseudomonas palustris, the inorganic salt NaHCO3 is used as carbon source, which is decomposed into CO2 under the action of carbonic anhydrase. Thiosulfate provides electrons. Part of CO2 is involved in cell growth and metabolism through Calvin Benson Basham cycle. Part of CO2 and electrons are synthesized into CH4 under the action of nitrogenase [8].
Finally, the Rhodopseudomonas palustris can use a variety of aromatic compounds as a separate carbon source. Lignocellulose hydrolysate is the energy source of microbial cell growth, but there are a lot of aromatic compounds in the hydrolysate, which can inhibit the growth of microbial cells. There is benzoyl COA pathway in Rhodopseudomonas palustris, which can decompose and metabolize many aromatic compounds [15-17]. At present, there are many studies on the aromatic compound kumarate. Kumarate is a kind of lignin monomer. When it is used as a carbon source, the nitrogen fixation efficiency and H2 production rate of Rhodopseudomonas palustris are relatively high, which are 2-5 times of that of acetic acid as a carbon source [18].
Table 1 available carbon sources of Rhodopseudomonas palustris
Table 1 Carbon sources utilized by R.palustris
To sum up, due to the wide variety of carbon sources available to Rhodopseudomonas palustris, and many carbon sources are relatively difficult to remove in industrial and agricultural waste water and waste gas treatment, therefore, from the perspective of carbon source utilization, Rhodopseudomonas palustris has great advantages as a microbial cell factory.
2. Gene engineering strategy: as a microbial cell factory, it is very necessary to knock out and express genes through gene engineering strategy. However, there is not much research on gene editing tools of Rhodopseudomonas palustris. In order to facilitate gene manipulation, inui et al. [25] screened pmg101 (15 KB) from 400 bacteria, which could be replicated in purple non sulfur bacteria. It was found that pmg101 was originated from Rhodopseudomonas palustris. Based on pmg101, pmg103 (5.68 KB) and pmg105 (5.68 KB) were obtained KB); under the condition of non selective pressure, these plasmids can stably replicate and propagate in Rhodopseudomonas palustris [25]. Xu et al. [26] overexpressed the genes of crtE, hpnd and DXS in Rhodopseudomonas palustris with plasmid pmg103 as the vector, so as to increase the production of squalene to 112 times of that of wild-type strains, reaching 15.8 mg / g.
Another shuttle plasmid, pbbr1mcs-5, can be used for vector construction and gene expression in Rhodopseudomonas palustris. Huang et al. [27] cloned the fix gene cluster into the polyclonal site of pbbr1mcs-5 plasmid, transformed the recombinant plasmid into Rhodopseudomonas palustris, and expressed the fix gene cluster in foreign countries. In addition to pbbr1mcs-5, pbbr1mcs-1, pbbr1mcs-2, pbbr1mcs-3, pbbr1mcs-4 and pbbr1mcs-4 of pbbr1mcs series Mcs_start can be applied to Rhodopseudomonas palustris, and can be selected according to actual needs [28].
In addition to gene expression, the study of gene knockout and gene replacement by suicide plasmid has also been reported in Rhodopseudomonas palustris. Pjq200ks is a kind of suicide plasmid widely used in Gram-negative bacteria. It contains p15A for plasmid replication and gentamicin resistance screening. It contains sacB gene (encoding sucrose-6-fructosyltransferase) from Bacillus subtilis, which can catalyze the hydrolysis of cane sugar to high molecular fructan and has lethal effect on Gram-negative bacteria [29]. Rhodopseudomonas palustris is a kind of Gram-negative bacteria. Rey et al. [18] used the suicide plasmid pjq200ks to transfer the mutated fragments of nifA * 571 and nifA * 574 into the chromosome of Rhodopseudomonas palustris to study the H2 production function of nitrogenase. In addition, the mutant nifdv75ah201q was transferred to the chromosome by pjq200ks to study the function of nitrogenase methane production [8].
In a word, there are few gene editing tools for Rhodopseudomonas palustris at present. In order to better apply Rhodopseudomonas palustris to the construction of microbial cell factories, it is necessary and urgent to study and explore its gene editing tools.
3. The application of Rhodopseudomonas palustris as a microbial cell factory 3.1 using Rhodopseudomonas palustris to produce gas fuel at present, human society has a great dependence on fossil fuels such as oil and coal, fossil energy is not renewable, and its production and application process causes great environmental pollution, so it is urgent to find clean energy.
The H2 production ability of Rhodopseudomonas palustris has been widely studied. In Rhodopseudomonas palustris, nitrogenase can transform N2 into NH3, and produce H2 at the same time. Moreover, in the absence of N2, nitrogenase can also catalyze the synthesis of H2, as shown in formula (1) [7]. Among them, the synthesis process of H2: the organic or inorganic substrate provides electrons, which are transferred to the ferritin through the photosynthetic electron transfer chain, and the light energy is converted to ATP through the photosynthetic electron transfer chain. ATP and the ferritin with electrons generate H2 under the action of nitrogenase [7]. Different from algae, the photosynthetic system of Rhodopseudomonas palustris can not decompose H2O, so it does not produce O2, which is more conducive to the production of H2. This photosynthetic bacteria can produce H2 faster [7,30].
Natural nitrogenase uses 75% of ferriredoxin to immobilize N2, resulting in low efficiency of H2 synthesis [18]. In addition, another product NH3 can inhibit the activity of nitrogenase at the transcription level and post translation level [31]. Rey et al. [18] obtained mutant strains through screening, which caused point mutation of transcription regulatory factors of nitrogenase subfamily nifA, so as to weaken the inhibition of NH3 on nitrogenase activity, and ultimately improve the production of H2. At present, the research on H 2 production of Rhodopseudomonas palustris mainly focuses on the investigation of different light intensity, different carbon sources, etc., but there are few reports on the improvement of H 2 production at the gene level, which may be due to the unclear metabolic pathway of H 2 production