Abstract
Our project aims to efficiently and massively synthesize the biofuel butyl butyrate through microbial fermentation, addressing energy-related issues on a global scale. To achieve this, we genetically engineered Clostridium tyrobutyricum (C. tyrobutyricum) to enable efficient and large-scale synthesis of butyrate and butanol. We employed a method of lipase catalyzed esterification of butyrate and butanol into butyl butyrate in which reaction the optimal butyrate-to-butanol molar ratio is 1:1. In the process of constructing C. tyrobutyricum, we used the method developed by Worldshaper-NJBIOX team last year which utilized the strain’s natural pathway for butyrate synthesis and introduced adhE2 to establish a butanol synthesis pathway. We further engineered C. tyrobutyricum to efficiently produce butyrate and butanol for the successive esterification into butyl butyrate through two strategies, adjusting the product molar ratio closer to 1:1 (butyrate:butanol) and co-enhancing the two products yield. The first strategy was achieved by enhancing the expression of deacetylase (Dac) in the synthesis pathway of butyrate and butanol, overexpressing CoA transferase (cat1) to inhibit the competing pathway of the byproduct, acetate, and using a weaker promoter Ptkt to express adhE2. The second strategy was fulfilled by overexpressing rate-limiting enzymes (bcd and crt) in the synthesis pathway. This allowed us to develop a C. tyrobutyricum strain with a high metabolic flux for the synthesis of butyrate and butanol and a more optimal product ratio, which can be used for efficient large-scale microbial fermentation for butyl butyrate.1. Project Goal
Our team is dedicated to address energy-related issues globally. We propose to efficiently and massively synthesize the biofuel butyl butyrate through microbial fermentation. Butyl butyrate can substitute traditional fossil fuels like petroleum and natural gas, thereby mitigating reliance on fossil fuel resources and boosting national energy security by reducing dependence on imported petroleum and natural gas. Additionally, the combustion of butyl butyrate generally results in lower greenhouse gas emissions. Utilizing microbial fermentation to synthesize butyl butyrate allows the use of renewable resources such as crop residues and vegetable oils as feedstock. This contributes to the advancement of renewable energy.
Our project was based on the project of Worldshaper-NJBIOX team last year. They utilized the natural pathway of C. tyrobutyricum for butyrate synthesis and introduced adhE2 to establish a butanol synthesis pathway. Based on this strain, we further engineered C. tyrobutyricum to efficiently produce butyrate and butanol with a better product ratio (closer to 1:1) for the successive esterification of these two precursors into butyl butyrate.
2. Project Overview
Our project to synthesize butyl butyrate via microbial fermentation is achieved in two steps.Step 1: Genetic engineering of C. tyrobutyricum
The first step is to genetically modify C. tyrobutyricum to synthesize butanol and butyrate effectively and with a favorable product ratio. We improved the expression of rate-limiting enzymes (bcd and crt) to co-enhance the production of butanol and butyrate. For the final esterification of butyl butyrate, the best butyrate-to-butanol molar ratio is 1:1. By introducing adhE2 into C. tyrobutyricum, the yield of butanol was much more than the butyrate yield. We found three approaches to adjust the product ratio to be as close to 1:1 as possible: (1) enhance Dac expression; (2) overexpress Cat1; (3) use a weaker promoter Ptkt instead of Pthl
This step is done by fulfilling five targets:
Target 1: Construct a butanol synthesis pathway.
Target 2: Overexpress Dac to directly enhance the synthesis pathway of butyrate and butanol through improving deacetylation.
Target 3: Overexpress Cat1 to indirectly enhance the synthesis pathway of butyrate and butanol by inhibiting the competing pathway which synthesizes by-product acetate
Target 4: Overexpress bcd and crt to directly enhance the synthesis pathway of butyrate and butanol through overexpressing rate-limiting enzymes.
Target 5: Use Ptkt promoter to express adhE2 to decrease butanol synthesis and indirectly increase butyrate synthesis
Step 2： Esterification reaction via lipase
The second step is to use the butanol and butyrate synthesized in step 1 to efficiently synthesize butyl butyrate via lipase catalyzed esterification reaction.
Fig. 1 Flow chart of our project to synthesize butyl butyrate using microbial fermentation
2. Project Overview
Step 1: Genetic engineering of C. tyrobutyricum
Target 1: Construction of a butanol synthesis pathway (dehydrogenase, adhE2) based on the project of Worldshaper-NJBIOX team
By comparing the native butanol synthesis pathway of Clostridium acetobutylicum and the butyrate synthesis pathway of C. tyrobutyricum, it was found that alcohol/aldehyde bifunctional dehydrogenase encoded by adhE2 gene can be used to construct a butanol synthesis pathway in C. tyrobutyricum [1]. This enzyme catalyzes the conversion of butyryl-CoA to butyraldehyde and then to butanol. Butyryl-CoA is an intermediate in the butyrate synthesis pathway in C. tyrobutyricum. By introducing adhE2, we can convert this intermediate into butanol (Fig.2). 2022 Worldshaper-NJBIOX team has engineered C. tyrobutyricum to express adhE2 using Pthl-adhE2 (BBa_K4408008) for the production of butanol (Fig.3). Our team used the same method to construct a butanol synthesis pathway in C. tyrobutyricum.
Fig.2 The native butyrate synthesis pathway and the proposed butanol synthesis pathway in C. tyrobutyricum
Fig.3 Genetic circuit of Pthl-adhE2
Target 2: Enhancement of butyrate and butanol synthesis (deacetylase, Dac)
Dac gene encodes a NAD-dependent deacetylase which removes acetyl groups from the N-terminal of protein substrates and weakening the N-terminal acetylation of proteins. Protein acetylation is the process of transferring the acetyl group from acetyl coenzyme A (Ac-CoA) to a specific site on a polypeptide chain and is one of the most common post-translational modifications (PTMs) in bacteria. The interplay between acetylation and deacetylation is crucial in the regulation of protein function. In C. tyrobutyricum, glucose is converted to acetyl-CoA which is mainly converted to butyryl-CoA and then to butyrate by enzymes of thiolase (thl), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), and butyryl-CoA dehydrogenase (bcd) (Fig.2); a part of acetyl-CoA is also converted to acetyl~P and then to a by-product acetate by enzymes coded by pta and ack[1]. In Clostridium acetobutylicum, by sensing the level of a series of intermediates in the acid/solvent synthesis pathway, an interplay between acetylation and deacetylation is enrolled to regulate the functions of the enzymes including those coded by pta, ack, thl, hbd, crt and bcd, in order to eliminate the accumulation of intermediates and finely regulate the outputs of the metabolic fluxes[2]. Since Clostridium acetobutylicum is closely related to C. tyrobutyricum, and pta, ack, thl, hbd, crt and bcd also code crucial enzymes in the butyrate/butanol synthesis pathway, we propose that in C. tyrobutyricum, acetylation and deacetylation are also important in reducing intermediate accumulation and controlling the carbon flow to different products such as butyrate and acetate by regulating the activities of these enzymes. Dac is a native gene in C. tyrobutyricum L319 coding a deacetylase which plays an essential role in deacetylation. We overexpressed Dac in C. tyrobutyricum to enhance such acetylation and deacetylation interplay in the pathway to improve the efficiency of butyrate and butanol synthesis.
Dac is expressed natively in C. tyrobutyricum L319. In this project, we used this native Dac gene to construct the recombinant plasmid of pMTL-Pthl-adhE2-Dac to overexpress Dac in C. tyrobutyricum (Fig.4). By enhancing the expression of deacetylase, we can increase the yields of butyrate and butanol directly.
Fig.4 Genetic circuit of pMTL-Pthl-adhE2-Dac
Target 3: Inhibition of the competing pathway: acetate synthesis (CoA transferase, cat1)
In C. tyrobutyricum L319, glucose is converted to pyruvate and subsequently to acetyl-CoA. Then, one part of acetyl-CoA is converted to acetate by phosphotransacetylase (pta) and acetate kinase (ack), another part is converted to ethanol, and the rest is converted into butyryl-CoA by several enzymes (Fig.2). Butyryl-CoA is respectively converted natively to butyrate by butyryl-CoA/acetate CoA transferase (cat1). Interestingly, this CoA transferase also converts acetate back to acetyl-CoA. In this way, CoA transferase reduces the acetyl-CoA to acetate flux and enhances the acetyl-CoA to butyrate flux. Therefore, the higher the expression of CoA transferase, the higher the butyrate/acetate ratio in the final product [1,3]. As mentioned in Target 1, we constructed a synthesis pathway of butanol from butyryl-CoA by introducing dehydrogenase (adhE2). So, the suppression of the acetyl-CoA to acetate flux also enhances the production of butanol.
The endogenous cat1 gene of C. tyrobutyricum L319 codes the butyryl-CoA/acetate CoA transferase. Using the cat1 gene and the native Pcat1 promoter of this gene, we constructed the part of Pcat1-cat1, and the recombinant plasmid of pMTL-Pthl-adhE2-Pcat1-cat1 to overexpress cat1 in C. tyrobutyricum (Fig.5), in order to suppress the acetyl-CoA conversion to acetate and indirectly enhance the metabolic flux of butyrate and butanol synthesis.
Fig.5 Genetic circuit of pMTL-Pthl-adhE2-Pcat1-cat1
Target 4: Enhancement of butyrate and butanol synthesis pathway (rate-limiting enzymes, crt and bcd)
In C. tyrobutyricum L319, glucose is converted to acetyl-CoA which is then mainly converted to butyryl-CoA by the following enzymes: thiolase (thl), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), and butyryl-CoA dehydrogenase (bcd) (Fig.2) [1]. Butyryl-CoA is subsequently converted to butyrate in the native strain and converted to butanol in the engineered strain in Target 1. In the preliminary experiment, we discovered that the yield of butyrate and butanol increased efficiently through the overexpression of bcd and crt. Therefore, crotonase and butyryl-CoA dehydrogenase coded by bcd and crt are considered as rate-limiting enzymes.
Here, we used the endogenous bcd and crt genes and Pthl promoter from C. tyrobutyricum L319 to construct the recombinant plasmid of pMTL-Pthl-adhE2-bcd-crt to overexpress crt and bcd in C. tyrobutyricum (Fig.6).
Fig.6 Genetic circuit of pMTL-Pthl-adhE2-bcd-crt
Target 5: Use Ptkt promoter to express adhE2 to decrease butanol synthesis and indirectly increase butyrate synthesis
During our project, we were inspired by a new part Ptkt developed by the Nanjing-BioX team. Ptkt is a native promoter that drives the expression of transketolase (tkt) gene in C. tyrobutyricum. Ptkt promoter was found to have weaker transcriptional strength than Pthl promoter. Since adhE2 expression driven by Pthl in our engineered strain showed too much exceeding synthesis of butanol to butyrate, we decided to replace Pthl with the weaker promoter Ptkt to obtain an engineered strain with less production of butanol and more yield of butyrate. So we constructed the recombinant plasmid of pMTL-Ptkt-adhE2 to engineer C. tyrobutyricum with a more optimal butyrate-to-butanol product molar ratio (closer to 1:1) for the esterification of butyl butyrate (Fig.7).
Fig. 7 Genetic circuit of recombinant plasmid pMTL-Ptkt-adhE2
Verification for Step 1
Colony PCR and DNA sequencing are used to assess the constructed recombinant plasmids in step 1. Verified plasmids are transferred to C. tyrobutyricum.
SDS-PAGE is used to verify protein expression in the strain.
To verify the function of genes transfected, we fermented the engineered strain utilizing glucose as substrate, and assessed the growth. The yields of butanol and butyrate were evaluated by HPLC.
Step 2： Esterification reaction for butyl butyrate synthesis via lipase
Lipase: CALB
Lipase-catalyzed esterification is a process where lipase is used to catalyze the reaction of alcohol and carboxylic acid to generate ester compounds. Butyl butyrate can be manufactured through the esterification of butyrate and butanol by lipases[5]. Candida antarctica lipase B (CALB) is an efficient biocatalyst commonly used in esterification. It is derived from Candida antarctica, one of the most prominent lipase producers among yeast[6].
Verification of Esterification
Gas chromatography was used to measure the yield of butyl butyrate from the esterification by CALB.
