Before any design started......
Why choose γ-PGA?
The purpose of our project is to treat saline-alkali soil containing salt and alkali, and to prevent secondary salinization, resist the infiltration of external salt water and interact with other organism. We need a substance that can reach all of these goals. It needs to have hydrogen ion, absorb exchangeable sodium ion, form soil aggregate and benefits plants.
So we find γ-PGA, a polymer formed by glutamic acid, which is a water-retaining agent that can satisfy our needs and is safe and green.
Fig.1 The structure of γ-PGA
In dealing with alkaline soil, the carboxyl group of γ-PGA can be ionized to neutralize the base(Wang et al.,2021).
In dealing with saline soil, γ-PGA adsorbed exchangeable sodium to its own body, so that the salt could be separated from soil colloid and washed to a deeper depth. It can also be coupled with calcium and magnesium ions in the soil. For chloride ions, γ-PGA on the surface of the soil can make chloride ions escape from the soil surface due to the electronegativity. In conclusion, γ-PGA allows the salts that form saline-alkali soils to go to depths that do not affect the topsoil.(Chen et al., 2021; Tang, 2015; Wen et al., 2019; Tan, 2018).
As a polymer formed by glutamic acid, γ-PGA has many hydroxyl and amino groups and large specific surface area, making it can effectively promote the formation of soil aggregates and preserve water evaporation. Soil aggregates increase soil porosity and are beneficial to shallow soil leaching. Because the main chain of γ-PGA contains a large number of hydrophilic carboxyl and peptide bonds, it can also slow water infiltration and inhibit surface water evaporation. It improves soil structure and prevents the return of salt water from deeper soils to the surface through capillary action, thus preventing secondary salinization.(Chen et al., 2021; Zhang, 2018; Wen, 2019; Hou, 2017)
In addition, γ-PGA can activate some ions, making nutrients easier absorbed by plants. It inhibits the crystallization of phosphate and increases the content of active phosphate. It can promote the absorption of N in soil, improve the absorption of P and K by plants, and reduce the loss of fertility. It also improves the biomass and activity around the roots, enhancing plant nutrient absorption capacity. γ-PGA improves the adaptability of soil plants and microorganisms to the environment. In this way, the plants can help to improve the saline-alkali land themselves.(Chen et al., 2021; Xu et al., 2014; Zhang et al., 2017)
γ-PGA is a component of natto, which is widely used in food, medicine, cosmetics and other industries, which is non-toxic. Moreover, γ-PGA can degrade slowly in the soil, and the degradation product is safe glutamate. It not only does not harm the soil, but is beneficial to the soil. So we do not need to recycle the γ-PGA produced by our project from the soil.
Overall, we choose the efficient and green γ-PGA for our project.
Why choose Corynebacterium glutamicum as our chassis?
When it comes to γ-PGA, there’s two kind of bacteria, Bacillus subtilis and Corynebacterium glutamicum. Bacillus subtilis needs the addition of glutamic acid, while Corynebacterium glutamicum can produce Glu itself. Besides, they both have a high tolerance of high salt concentration and pH.
We were thinking of using a symbiotic system of Bacillus subtilis and Corynebacterium glutamate. However, during further discussion with experts, our team's instructor Yang Jinshui pointed out that these two bacteria are not known mutually beneficial symbiotic relationship in soil, and if they are isolated in space, the treatment efficiency will be greatly reduced. Therefore, we decided to make our dream come true by one chassis alone.
There is only a few studies on Bacillus subtilis synthesizing Glu itself, but most people use mediums that have Glu to cultivate Bacillus subtilis, in order to produce γ-PGA.(Nguyen et al., 2018; Sib Sankar Giri et al., 2015)
As for Corynebacterium glutamicum producing γ-PGA, however, has already had plenty of solid researches, and even 2019_JNU_China has had involved in rising the yield of γ-PGA by Corynebacterium glutamicum as well.(Hsueh et al., 2017; Liu et al., 2017)
Moreover, related studies and molecular cloning materials for Corynebacterium glutamicum expression of exogenous genes from Bacillus subtilis are also well developed.
What’s more, Corynebacterium glutamicum inhabit in the soil originally, which means it adapts to the living in it pretty well. In addition, lots of research show that Corynebacterium glutamicum has a strong tolerance to both salty and alkali environment. They can grow in pH 9.0 without any influence on its metabolism and live in 1.0M NaCl for at least 12 days, which indicate that Corynebacterium glutamicum is suitable for saline-alkali soil amelioration.(Xu et al., 2018; Follmann et al., 2009)
Thus, we choose Corynebacterium glutamicum as our final and only chassis.
Goal 1: Make Corynebacterium glutamicum able to synthesize γ-polyglutamic acid
In our design, we need to transfer related genes into Corynebacterium glutamicum to endow it with the capacity to produce γ-PGA. Synthetic genes for γ-PGA in Bacillus subtilis or other Bacillus spp. are capA, capB, capC, capE (or pgsA, pgsB, pgsC, pgsE). This is the cluster of genes present in Bacillus subtilis and related species, which is responsible for the synthesis of γ-polyglutamic acid. Moreover, previous studies have found that these four genes are not all necessary for the production of γ-PGA. The prevailing view is that only three of these genes, capA, capB, capC (or pgsA, pgsB, pgsC), are required to perform their entire function(Ashiuchi, Soda et al. 1999, Cao, Geng et al. 2013). Among these three, capB and capC can use glutamate to produce γ-PGA, and capA is responsible for the transportation of γ-PGA to the extracellular compartment.
Fig.2 How capA, capB and capC work together
(A: protein product of capA,B: protein product of capB,C: protein product of capC)
Based on previous studies, we use Corynebacterium glutamicum ATCC13032 as our chassis organism for γ-PGA production. Three genes, capA capB capC, are amplified from Bacillus subtilis str. 168 using PCR, ligated using Gibson Assembly technique and transformed into Corynebacterium glutamicum ATCC13032. The endogenous constitutive strong promoter P0864 of Corynebacterium glutamicum is selected as the promoter of the three genes, because our final aim is to put our bacteria into the soil to restore it, where no induction can be executed. Due to the excellent ability of Corynebacterium glutamicum to synthesize glutamate, we do not need to add additional glutamate as feedstock. Eventually, Corynebacterium glutamicum will produce a certain amount of γ-polyglutamic-acid polymerized from glutamic acid in vivo. Due to the function of capA to transport γ-PGA to the extracellular compartment and the nature of the transmembrane structure itself, polyglutamic acid will not be blocked in the bacteria.
Fig.3 Genetic circuit for γ-PGA synthesis
Goal 2: Make Corynebacterium glutamicum able to synthesize γ-PGA at high yield
In order to promote our engineered Corynebacterium glutamicum strain’s ability of γ-PGA production, we decide to further construct it into a glutamic acid high-yielding strain. Since glutamic acid is the only substrate for γ-PGA synthesis, we believe that the idea of such construction is worth a shot.
It is known that glutamic acid is formed from the citric acid cycle intermediate α-ketoglutarate, therefore proper regulation of the TCA cycle can help us achieve our goals. Furthermore, the oxaloacetate node and α-ketoglutarate node play important roles in the TCA cycle, so we will perform our work there. For the oxaloacetate node, overexpressing ppc, which encodes PEPC that catalyzes PEP to form pyruvate will introduce more carbon fluxes to the TCA cycle, and finally resulting a gain in glutamic acid yield. And for the α-ketoglutarate node, attenuating odhA, which encodes KGDH that catalyzes α-KG to form succinyl-CoA will introduce more carbon fluxes to glutamic acid synthesis rather than to the succinyl-CoA formation. Previous studies have shown that this kind of modification can result in more glutamic acid indeed and does little harm to bacteria's TCA cycle.
Fig.4 The TCA cycle (Qiao, Z. et al. 2020)
For ppc overexpression, we introduce a stronger strength RBS to the upstream of its ORF(Qiao, Z. et al. 2020), thus increasing ppc translation.
Fig.5 Genetic circuit for ppc overexpression
As for odhA attenuation, we have two thoughts. One is to replace the original RBS with a lower strength one to attenuate odhA translation(Zhang, B. et al. 2017). The other is through RNAi, which is designed by introducing a weak promoter to the upstream of the antisense strand of odhA to generate the possibility for the antisense RNA of odhA to transcript. And hopefully, a certain amount of the antisense transcripts will form complementary duplexes with the mRNA of odhA(Kim, Hirasawa et al. 2009), therefore, to some extent block odhA translation. With this strategy, we can knockdown odhA, meanwhile, reserving some of its expression since odhA is rather crucial in the TCA cycle, and a complete knockout would certainly inhibit the growth of bacteria. What is more, hence that all the modifications described above will be inserted into the genome of the target strain through pK18mobsacB, a mobilizable vector with KmR and sacB allowing for selection of double crossover in C. glutamicum(Zhang, B. et al. 2017). Last but not least, after the genetic modification, assays for activity of the related enzymes and glutamic acid yield will be conducted in order to evaluate the effectiveness of each design, and the one with the best effect will be used in our final construction.
Fig.6 Genetic circuit for odhA knockdown by changing the RBS
Fig.7 Genetic circuit for odhA knockdown by RNAi
Goal 3: Biosafety —— Kill switch
Considering the application of our engineered bacteria, we should design a particular kill switch to ensure biosafety. The basis of our kill switch is to allow our bacteria to function in saline or alkaline soil, which means we need to maintain their survival at high ionic strength or high pH or both. When mission completed and the ionic strength and pH fall back to normal, they simply suicide to prevent impacting the natural bacteria in normal soil.
To achieve this, we use an salt-inducible promoter Pgsib, originally from Bacillus subtilis. PgsiB is recognized by sigma B, which is a transcription factor closely related to the response for challenging pressure and is only activated when pressure comes around. According to the literature(Maul, Völker et al. 1995), the addition of NaCl to a concentration of 4% can make the gene after PgsiB transcribe 111 times before the imposition of stress. On a protein level, researchers also have found that the salt stress can nearly double the flourescence when using gfp as marker gene(Promchai, Promdonkoy et al. 2016).
We also use an alkali-inducible promoter Patp2 from Corynebacterium glutamicum itself, which is regulated similarly by an adversity-response transcription factor sigmaH. When the bacteria grow at alkaline pH, the external pH of the bacteria triggers the change of internal pH, which then acts as an intracellular signal to make the the content of sigmaH factor increased. When the content of sigmaH increased, the binding efficiency with Patp2 promoter increased, and the expression of gene downstream of Patp2 promoter will boost as well(Barriuso‐Iglesias, Barreiro et al. 2013). Because of the enhanced response of alkali promoter Patp2 in alkaline environment, it is commonly used as a pH response element in synthetic biology(Gao, Xu et al. 2021).
Fig.8 Patp2 can respond to alkali stress(Barriuso‐Iglesias, Barreiro et al. 2013)
Besides, we use the gene ndoA from Bacillus subtilis which encodes a toxic component of a type Ⅱ toxin-antitoxin system that can kill bacteria by cleavaging the mRNA on specific sequence(Park, Yamaguchi et al. 2011). The toxicity of the product of ndoA in Bacillus subtilis and E.coli has already had experimental proof (Pellegrini, Mathy et al. 2005, Simanshu, Yamaguchi et al. 2013). We assume that it is toxic for the growth of Corynebacterium glutamicum as well and will do relevant verification experiments.
Finally, we introduce the lac operon to help our kill switch realize. The repressing gene lacIq and the promoter Ptac come from plasmid pXMJ19, which is a shuttle vector and can ensure the availability of lac operon in Corynebacterium glutamicum(Jakoby, Ngouoto-Nkili et al. 1999).
Fig.9 Genetic circuit for our kill switch
To sum up, the logic for our kill switch is pretty like an "OR" gate. With the help of these parts, our kill switch will work in this way: at high saline or alkaline or both, the lacIq behind Pgsib and Patp2 will express and the repressor will bound to tac promoter to inhibit the expression of the toxin gene ndoA. At low saline and alkaline, there is no longer repression and ndoA will express, leading to the death of bacteria.
Plus: Inserting genes into genome
Since we plan to put the engineered bacteria into the natural environment without antibiotics screening pressure, the plasmid could be easily lost, making our engineered bacteria lose the function of synthesizing γ-PGA. So, after all the verification experiments, we want to insert our final genetic circuits into the plasmid pK18mobsacB, upstream and downstream with carefully selected homologous sequence to the genome of Corynebacterium glutamicum ATCC13032. Using the double screening features of the plasmid pK18mobsacB, we can select the engineered bacteria in which our genetic circuits have been inserted into its genome successfully(Zhang, et al. 2014).
Fig.10 The procedure for inserting sequence into the genome of bacteria using pK18mobsacB
 Ashiuchi, M., et al. (1999). "A poly-γ-glutamate synthetic system of Bacillus subtilis IFO 3336: gene cloning and biochemical analysis of poly-γ-glutamate produced by Escherichia coli clone cells." Biochemical and biophysical research communications 263(1): 6-12.
 Barriuso‐Iglesias, M., et al. (2013). "Transcriptional control of the F 0 F 1‐ATP synthase operon of Corynebacterium glutamicum: SigmaH factor binds to its promoter and regulates its expression at different pH values." Microbial biotechnology 6(2): 178-188.
 Cao, M., et al. (2013). "Engineering of recombinant Escherichia coli cells co‐expressing poly‐γ‐glutamic acid (γ‐PGA) synthetase and glutamate racemase for differential yielding of γ‐PGA." Microbial biotechnology 6(6): 675-684.
 Gao, X., et al. (2021). "Recent advances in engineering of microbial cell factories for intelligent pH regulation and tolerance." Biotechnology Journal: 2100151.
 Hsueh, Y.-H., et al. (2017). "Poly-γ-glutamic acid synthesis, gene regulation, phylogenetic relationships, and role in fermentation." International journal of molecular sciences 18(12): 2644.
 Jakoby, M., et al. (1999). "Construction and application of new Corynebacterium glutamicum vectors." Biotechnology Techniques 13(6): 437-441.
 Kim, J., et al. (2009). "Effect of odhA overexpression and odhA antisense RNA expression on Tween-40-triggered glutamate production by Corynebacterium glutamicum." Applied microbiology and biotechnology 81(6): 1097-1106.
 Lee, J.-Y., et al. (2016). "The actinobacterium Corynebacterium glutamicum, an industrial workhorse."
 Maul, B., et al. (1995). "σ B-dependent regulation of gsiB in response to multiple stimuli in Bacillus subtilis." Molecular and General Genetics MGG 248(1): 114-120.
 Park, J.-H., et al. (2011). "Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase." FEBS letters 585(15): 2526-2532.
 Pellegrini, O., et al. (2005). "The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor." Molecular microbiology 56(5): 1139-1148.
 Promchai, R., et al. (2016). "A novel salt-inducible vector for efficient expression and secretion of heterologous proteins in Bacillus subtilis." Journal of biotechnology 222: 86-93.
 Qiao, Z., et al. (2020). "Effect of key notes of TCA cycle on L-glutamate production." Sheng wu Gong Cheng xue bao= Chinese Journal of Biotechnology 36(10): 2113-2125.
 Simanshu, D. K., et al. (2013). "Structural basis of mRNA recognition and cleavage by toxin MazF and its regulation by antitoxin MazE in Bacillus subtilis." Molecular cell 52(3): 447-458.
 Zhang, B., et al. (2017). "Systematic pathway engineering of Corynebacterium glutamicum S9114 for L-ornithine production." Microbial cell factories 16(1): 1-10.
 Zhang, et al. (2014).A new plasmid for gene transformation of Corynebacterium glutamicum and its application. CN201310742881. X. 2014. 04. 09.
 Wang Ziyan et al. Mitigation of soil salinization and alkalization by bacterium-induced inhibition of evaporation and salt crystallization[J]. Science of the Total Environment, 2021, 755(P1).
 Chen L , Su W , Xiao J , et al. Poly-γ-glutamic acid bioproduct improves the coastal saline soil mainly by assisting nitrogen conservation during salt-leaching process[J]. Environmental Science and Pollution Research, 2021, 28(7):1-9.
 Tang Dong.Remediation of the Secondary Saline Soil by γ-polyglutamic Acid and Halophytes [D].Southwest University,2015.
 WEN Liun SHI Weniuan PANG Linna. Effects of Poly-γ-glutamic Acid on Soil Water Infiltration and Water and Salt Transport[J].Journal of Soil and Water Conservation, 2019,33(03):76-80+87.
 Tan Wenjuan.Effect of polyglutamic acid on evaporation and crystallization of salt in saline-alkali soil [D].University of Chinese Academy of Sciences,2018.
 Zongqi Xu,Peng Lei,Xiaohai Feng, et al.Calcium involved in the poly(γ-glutamic acid)-mediated promotion of Chinese cabbage nitrogen metabolism[J].Plant Physiology and Biochemistry,2014,80:144-152.
 Lei Z , Yang X , Gao D , et al. Effects of poly-γ-glutamic acid (γ-PGA) on plant growth and its distribution in a controlled plant-soil system[J]. Scientific Reports, 2017, 7(1):6090.
 Zhang Chen. Biosynthesis of Poly-y-glutamic Acid and Its Application to Soil Remediation and Improvement[J].Bulletin of Soil and Water Conservation, 2018,38(02):323-328.
 HOU Yaling ZHOU Beibei WANG Quanjiu CHEN Xiaopeng TAO Wanghai ZHANG Jihong.Effects of Bacillus Subtilis on Water Movement and Water Stable Aggregate in Saline Alkali Soil[J]. Journal of Soil and Water Conservation,2017,31(04):105-111+147.
 Haifeng Zhu Youdong Liu Shutang LI Kunlun Lu Zhenyu Zhao Lin. Effects of Water Retention Agent and Organic Acid Soil Conditioner on Saline-alkali Soil Improvement and Wheat Yield[J]. Shandong Agricultural Sciences,2020,52(04):121-125.
 Nguyen S , Inaoka T , Kimura K . Poly-γ-glutamic Acid Production by Bacillus subtilis (natto) under High Salt Conditions[J]. Japan Agricultural Research Quarterly, 2018, 52(3):249-253.
 D, Sib Sankar Giri A , et al. "Production and characterization of a thermostable bioflocculant from Bacillus subtilis F9, isolated from wastewater sludge." Ecotoxicology and Environmental Safety 121(2015):45-50.
 Shih IL, Van YT. The production of poly-(gamma-glutamic acid) from microorganisms and its various applications. Bioresour Technol. 2001 Sep;79(3):207-25. doi: 10.1016/s0960-8524(01)00074-8. PMID: 11499575.
 Hsueh YH, Huang KY, Kunene SC, Lee TY. Poly-γ-glutamic Acid Synthesis, Gene Regulation, Phylogenetic Relationships, and Role in Fermentation. Int J Mol Sci. 2017 Dec 7;18(12):2644. doi: 10.3390/ijms18122644. PMID: 29215550; PMCID: PMC5751247.
 Liu X, Zhao Z, Zhang W, Sun Y, Yang Y, Bai Z. Bicistronic expression strategy for high-level expression of recombinant proteins in Corynebacterium glutamicum. Eng Life Sci. 2017 Aug 14;17(10):1118-1125. doi: 10.1002/elsc.201700087. PMID: 32624739; PMCID: PMC6999366.
 Xu N , Zheng Y , Wang X , et al. The lysine-299 residue endows multi-subunit Mrp1 antiporter with dominant roles in Na+-resistance and pH homeostasis in Corynebacterium glutamicum[J]. Applied and Environmental Microbiology, 2018:AEM.00110-18.
 Follmann, M., Ochrombel, I., Krämer, R. et al. Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis. BMC Genomics 10, 621 (2009).