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Library of functional acids


In our project, the regulation sub-system is constructed to adjust the microenvironment pH by synthesis of acid and alkali. These chemicals can be secreted outside the bacteria and thus change the microenvironment pH. A library for acid synthesis is established and the following kinds of acid are included: lactic acid, formic acid, acetic acid, succinate, 3-hydroxypropionic acid and 3-hydroxybutyrate. Therefore, we can apply different genes in the regulation sub-system according to various situations or wide-ranging needs.


  • ○ Lactic acid[Expand]

      L-Lactic acid, one of the most important chiral molecules and organic acids, is produced via pyruvate from carbohydrates in diverse microorganisms catalyzed by an NAD+-dependent L-lactate dehydrogenase [1]. The ldhA gene is used in the synthesis of lactic acid. This gene encodes an L-lactate dehydrogenase (L-LDH) which converts pyruvate to L-lactate [2]. The simple pathway of lactic acid synthesis is shown in the figure below:


  • ○ Formic acid[Expand]

      Formic acid is a signature fermentation product of many obligate and facultative anaerobes [3-5]. One core enzyme, pyruvate formate-lyase 1[6], is applied to the synthesis of Formic acid. This enzyme work only under anaerobic condition. The figure below shows a simple pathway of formic acid synthesis:



  • ○ Acetic acid[Expand]

      In synthesis of acetic acid, two enzymes, phosphate acetyltransferase (pta) [7] and acetate kinase A (ackA) [8] are used. Phosphate acetyltransferase is an enzyme that catalyzes the chemical reaction:

      Substrates of this enzyme are acetyl-CoA and phosphate, whereas its two products are CoA and acetyl phosphate [9-11]. ackA can facilitate the production of acetyl-CoA by phosphorylating acetate in the presence of ATP and a divalent cation[12-14]. The brief pathway of acetic acid synthesis is shown in the figure below.


  • ○ Succinic acid[Expand]

      Succinate has been recognized as an important platform chemical that can be produced from biomass. [15] There are two main methods in fermentation to synthesize succinate(Fig.1,2). One is under aerobic condition and the other is under anaerobic situation. Gene silence technology [16] can be employed in aerobic pathway. Genes of PoxB, Pta, sdhA and some other enzymes could be knocked out to ensure a high succinic acid yield [15].



      Fig.1 Anaerobic pathway



      Fig.2 Shikimic acid pathway



  • ○ 3-hydroxypropionic acid[Expand]

      3-Hydroxypropionic acid (3-HP) is an important platform chemical from which several commodity and specialty chemicals can be generated[17]. DhaB, a coenzyme B12-dependent glycerol dehydratase, converts glycerol to 3-hydroxypropionaldehyde (3-HPA). GdrAB is a glycerol dehydratase-reactivating factor [18]. GabD4, a novel aldehyde dehydrogenase, is responsible for the oxidation of aldehydes to carboxylic acids [19]. The brief 3-hydroxypropionic acid synthesis pathway is shown below [20]



  • ○ 3-hydroxybutyric acid[Expand]

      Poly (3-hydroxybutyrate) (P(3-HB)) is a polyester of 3-hydroxybutyrate that is synthesized by a variety of microorganisms as intracellular carbon and energy storages under unbalanced nutrients conditions [21-22]. An operon of three genes (organized as phaCAB) encodes the essential proteins for the production of P(3-HB) in the native producer, Ralstonia eutropha [23]. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase.

      The synthesis pathway of 3-hydroxybutyrate is shown below.





  • ○ Shikimic acid[Expand]

      Shikimic acid is a highly functionalised, six-carbon ring with three chiral carbons and a carboxylic acid functional group [24]. Shikimic acid pathway exists naturally in microorganisms and therefore they can be employed to overproduce this acid from carbon sources such as glucose. The Shikimic acid pathway is shown below(Fig.3).


      Fig.3 Shikimic acid pathway



○ Results

There are many kinds of acid in our acid synthesis parts, we don’t have enough time to test them one by one. So we chose gene LdhA, a gene coding L-lactate dehydrogenase and product L-lactic acid, as our model gene to test in our whole system.

To verified the LdhA gene, we constructed our gene into pET28a (Fig.4,5), and induced its over-expression by IPTG (Isopropyl β-D-1-Thiogalactopyranoside) under 16 ℃ in BL21(DE)3.

Fig.4 The plasmid pET28a and gene LdhA

Fig.5 The constructed result of LdhA-pET28a


Meanwhile, we put glucose as the substrate of glycolysis (40% glucose solution, add 2ml to 48ml culture solution). After 12 hours fermentation, we measured the pH of the culture fluid. We use the pET28a(BL21(DE3)) as the control. We repeated this process three times and our result is shown in Table.1.


Table.1 The pH value of culture fluid

Group pH value(Three times) pH value(average)
pET28a(Aerobic condition) 7.40 7.45
7.53
7.42
pET28a(Anaerobic condition) 6.28 6.25
6.22
6.25
LdhA(Aerobic condition) 7.09 7.15
7.18
7.18
LdhA( Anaerobic condition ) 5.25 5.27
5.30
5.26

According to the Table.1, we could easily found that there is an obvious difference between experimental group and control. Under the anaerobic condition, L-lactate dehydrogenase, coding by LdhA, acts much better ability than under aerobic condition. However, the final pH value of culture fluid is bigger than pKa of lactic acid. We suppose that the bacteria take advantage of lactic acid, due to the lack of carbon source.


○ Conclusion

There are too many kinds of acid, so we didn’t have enough time to verify them. However, we chose LdhA, coding lactate dehydrogenase which converts pyruvate to lactate, as our model gene to test in our whole system. According to the result, we could confirm that our acid production part is working. However, it’s not difficult to find that most of acid production genes are related to oxidative respiratory chain, and others verified them through gene silence. So we prefer to introduce CRISPR/Cas to make this part work better.The summary of acid synthesis library is shown below(Table.2).


Table.2 The summary of acid synthesis library

Kinds of acid Gene used Acidity strength
(Pka1)
Lactic acid ldhA 3.86
Formic acid pflB 3.75
Acetic acid Pta＆ackA 4.76
Succinic acid
(Aerobic pathway)		 \ 4.21
Succinic acid
(Anaerobic pathway)		 \ 4.21
3-hydroxypropionic acid DhaB 4.50
3-hydroxybutyric acid phaC, phaA and phaB \
Shikimic acid aroB, aroD~aroH 3.87±0.6

Error-Prone PCR


○ Background

Similar with Polymerase chain reaction(PCR), error-prone PCR is also a powerful method to produce linear DNA fragments, but results in random mutagenesis. This mutagenic PCR, which introduces random mutations by reducing the fidelity of the DNA polymerase[25]. This process is similar to standard PCR cloning of a target sequence except that, for constructing a diverse mutagenesis library, a much larger pool of transformants are needed to increase the diversity of the library, making the process tedious and inefficient[26]. As a result, various processes have been developed to streamline the methods.


○ Design

Step by step instructions(Fig. 6):
1. Amplify the target gene under error-prone condition.
2. Ligate the DNA fragment into a plasmid.
3. Transform the plasmid into a competent cell.
4. Pick out mutants shifting to 96-well plates for more experiments[27][28].

Fig.6 Four steps of EP-PCR


Conventionally, P-atp2 promoter wasn’t as well function as a mechanical device, and our machine could never use the only one alkali-induced promoter to regulate all the complex situation. What’s more, we anticipated knowing the accurate response range of P-atp2 promoter, so we could extend it. Thus, the idea was appearing, by using error-prone PCR to mutate promoter, which has different efficiency and different ranges of response to the environment.

The process of mutating P-atp2 is cockamamie, when it does not require excellent skill of experiment or advanced technology. Even though there are several ways to change the reaction condition, we finally chose to put manganese ion into our error-prone reaction system.

After the amplification, agarose gel electrophoresis analyzed the promoter DNA fragment, which was using the P-atp2 as the template of error-prone PCR. The size of P-atp2 is about 355bp. Then the right gel was extracted. And another agarose gel electrophoresis analysis was done, while the P-atp2 was linked to Lac Z alpha by using the OE-PCR. Then we ligated the production into pXMJ19 and transformed the plasmid into BMTOP10.

While the whole positive-bacteria couldn’t be active 100%, our promoters still needed to be verified.

In order to attain our goal, the strategy we have devised was measuring the activity of enzyme β-galactosidase(LacZ). Most recently, we picked out every other 94 bacteria of experiment groups into a 96-well cell culture plates, the rest of two wells were the negative control(NC) and the positive control(PC). As these bacteria grew normally and the OD600 was up to about 1.5, we lysed cells, then added ONPG to start a color reaction and recorded the response time. After the sodium carbonate was added to terminate the reaction, we measured the OD420 and OD550, which could be put into the formula to calculate the activity.(Table.3)

Table.3 the formula to calculate the activity of β-galactosidase

Enzyme Activity= 1000*(OD420-1.75*OD550)/(t*0.1*OD600)


○ Result

In the first round of experiments, we obtained over 1700 bacterial colonies, ultimately, there was about one thousand of them were positive-bacteria. In the second verification , we selected 9 typical bacteria whose promoter has strong activity.

Table.4

Promoter Enzyme Activity
pH=5 pH=6 pH=7 pH=8 pH=9
P-atp2-mutant-140 5481.93 2035.64 2265.33 3545.26 8650.00
P-atp2-mutant-154 1757.99 2181.08 1918.48 6786.56 5787.04
P-atp2-mutant-202 3333.33 2088.99 1335.43 1842.95 4730.26
P-atp2-mutant-226 3588.87 2659.88 1913.91 3011.70 5671.05
P-atp2-mutant-317 3225.00 3866.46 2764.05 8458.33 5229.01
P-atp2-mutant-399 4284.27 3282.19 4856.48 3938.55 6722.07
P-atp2-mutant-430 7388.89 5059.94 787.13 1055.39 969.83
P-atp2-mutant-705 1750.73 2395.83 1917.76 4949.71 19923.37
P-atp2-mutant-706 1062.38 1382.87 3642.15 9321.94 10627.89
Positive Control 1662.88 1892.42 2416.13 4065.80 3086.04


Recombinase System


○ Introduction

Recombinase, most of which come from bacteria and fungus, is a kind of enzyme that can make gene recombine [27][28]. It’s usually used to change the structure of genome, regulate the expression of gene and so on. A special sign site in genome (30-40bp) can be recognized by DNA recombinase, and the DNA recombinase will catalyze the recombination of the gene between two sign sites [29]. This ability allows the recombinase four basic functions: gene excision and insert, inversion, translocation and cassette exchange. These functions can be applied to edit the structure of genome, and also to regulate the expression of gene directly [30][31]. There are many kinds of recombinase, such as Cre, Flp, RecA/RAD51, Tre, Hin and others. Different kinds recombinase have different recognized site, so they wouldn’t interfere with other recombinases’ function.


○ Design

In order to fine regulate the pH of environment, we introduce the recombinase system here. We have two libraries of acid and alkali, when the different assembles of acid and alkali is used in our whole system, we need to use different methods to regulate it. So we designed three gene circuits using different kinds of recombinase to achieve the fine-regulation function.


1)Cre-LoxP and Flp-FRT circuits


Cre is a kind of Tyrosine recombinase (YR) from P1 bacteriophage. It can catalyze the recombination of the gene between the two specific DNA sign sites (LoxP site). The Cre-LoxP system consists of recombinase Cre and LoxP. When the two LoxP sites are in the same direction, the gene between them would be excised. However, if two sites are in the opposite direction, the gene would be inverted (Fig.7) [32]. Cre does not need ATP or other cofactor to achieve its function [33][34]. The length of LoxP is 34 bp, it contains two inverted repeat sequences (13 bp) and the interval sequence (8 bp). The interval sequence also can define the direction of LoxP(Fig.8).


Fig.7 The function of the recombinase


Fig.8 The sequence of LoxP site


Flp is the other kind of recombinase found in Saccharomyces cerevisiae. It can recognize the FRT sign site. Flp and FRT form the Flp-FRT system. The function of Flp-FRT system is the same as Cre-LoxP system’s. FRT site has the similar structure with LoxP site (Fig.9) [35].

Fig.9 The sequence of FRT site


Cre-LoxP system and Flp-FRT system can be used in Recombinase-mediated cassette exchange (RMCE) [36][37], so we use the principle of RMCE and design the first kind of our fine-regulation circuits (Fig.10).

Fig.10 The gene circuit of Cre/Flp-mediated recombinase circuits


In fermentation process, the environment usually turn acidic, so we produce alkaline substance(γ-aminobutyric acid, GABA) [38] in the primary state. And alkali-induced promoter P-atp2 and acid-induced promoter P-asr are employed to produce the recombinase Cre and Flp respectively. When the pH of environment reaches the P-atp2’s responsive range, Cre would express and recognize the LoxP sites at both sides of strong constitutive promoter J23119 and it would be inverted. Therefore the bacteria stop to produce lactate[39]. Similarly, when the pH of environment reaches the P-asr’s responsive range, J23119 would be inverted again. The bacteria will produce GABA again. Through the combination of multiple LoxP sites and FTR sites, we can realize multi-regulation and fine-regulation function.

To prevent gene from wrong inverting, we designed two kinds of sRNA to inhibit Cre and Flp’s function respectively. When one of recombinases expresses, the sRNA for the other recombinase will be produced at the same time. The sRNA would inhibit the translation of the other recombinase and we can avoid the wrong inversion. sRNA consists of two parts, the scaffold structure MicC and a target-binding sequence. Target-binding sequence, an anti-sequence about 15~20 bp, can inhibit the translation through binding with mRNA [40][41].

These circuits is used when the functional acid and the alkali are both medium.

2)Bxb1 circuit


The Bxb1 integrase is a DNA recombinase, more precisely a member of serine recombinase family. Bxb1 could be divided to two parts, gp35 and gp47. The gp35 part could recognize specific sequences, called attB and attP, and then integrate, invert, or excise dsDNA depending on the orientation of recognition sequences. When it inverts DNA sequence, the attB and attP sequences are changed into attL and attR, as other DNA recombinases do. Another part called Bxb1 gp47 binds to integrase-DNA complex and this complex flips DNA back into original sequence by regenerating attB and attP sequences. So the efficiency of inversion mediated by Bxb1 is half and half [42].

According to the function of Bxb1, we design the second type circuit to regulate the environmental pH(Fig.11)


Fig.11 The gene circuit of Bxb1-mediated recombinase system


This circuit is applied when the functional acid and the alkali are both strong. In the primary state, the bacteria also produce alkali. Since the alkali is strong, the pH of environment could reach the responsible range of P-atp2 easily. It can lead Bxb1 to work. The promoter J23119 could be inverted persistently until the inversion efficacy reaches half and half. In this process, the bacteria produce the acid and the alkali at the same time, it can create a wave-motion of pH. And the pH of environment will be stable finally. The final pH is influenced by the promoter sensing pH and the strength of acid and alkali.


3)FimE circuit


There is another condition. In this condition, we use the assemble of the strong acid and weak alkali, or strong alkali and weak acid. We use FimE to achieve the fine-regulation. This is our gene circuit of FimE-mediated recombinase system. (Fig.12)



Fig.12 The gene circuit of FimE-mediated recombinase system


FimE is different from other kind’s recombinase. Its recognization sites on the upstream (IRL) and downstream (IRR) are not same. It means that after the recombination mediated by FimE happens, the sign sites would be exchanged, so that the new sign sites couldn’t be recognized by FimE again. Thus the recombination mediated by FimE is irreversible.[43]

Granted that the bacteria produce the strong alkali in the primary, the environment would turn alkaline quickly. So P-atp2 can correspond the change of environment and start the expression of FimE. FimE recognizes IRL and IRR, then catalyze the inversion of J23119. The weak acid would be generated at that time. For the environment, due to the production of the weak acid, it would turn neutral gradually.

Since the recombinases were found and RMCE was invented, more recombinases’ functions and features were be explored. Recombinase has stick logic relationship and high robust. It fits the modern research of synthetic biology.

○ Result


To test and verify the recombinase system, we replace the mLdhA and gadA to mRFP and GFP respectively. (Fig.13) Through fluorescence, we could verify the recombinase system.

Fig.13 The verification circuits of recombinase system


We constructed our verification circuits through OE-PCR, and linked them into vector pSB1C3 (Fig.14~17). And we divided it into recombinase part and fluorescence part.

Fig.14 The constructed result of Bxb1, P-atp2 and P-atp2+B0034+Bxb1

(channel 1-3 are P-atp2, Bxb1 and P-atp2+B0034+Bxb1 respectively)


Fig.15 The constructed result of FimE, P-atp2 and P-atp2+B0034+FimE

(channel 1-3 are P-atp2, Bxb1 and P-atp2+B0034+Bxb1 respectively)


Fig.16 The construction result of FimE’s verification device K137058

(channel 1~6 the construction result of K137058, channel 1-3 and 6 are positive.)


Fig.17 The construction result of Bxb1’s verification device

(channel 1~6 the construction result of Bxb1’s verification device, channel 1-3 are positive.)


Meanwhile, we constructed the verification devices, fluorescence part, respectively, then co-transformed them into BMTOP10. We observed their fluorescent condition by fluorescence microscope and floe cytometry.

Fig.18 The fluorescence result of FimE’s verification device


Fig.19 The fluorescence result of Bxb1’s verification device


○ Conclusion


In the modern fermentation process, fine-regulation is of vital importance. So this year, in our project, we took advantage of recombinase system to come it true. Although we didn’t have enough time to verify all of them, according to the results of Bxb1 system and FimE system, the induced promoter would be inverted in the alkaline environment. Due to the logic relationship and high robust of recombinase, we believed that our recombinase system could realize the fine-regulate functions.



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