Our Design

Abstract

Our team aims to use kitchen waste, shrimp shells, as raw material to yield a biofuel, butyl butyrate, through the efficient biological production of engineered Clostridium tyrobutyricum (C. tyrobutyricum) and a catalyzed reaction by a lipase from engineered E. coli.

• 1) For the engineered C. tyrobutyricum, native C. tyrobutyricum contains a synthesis pathway of butyrate. An intermediate butyryl-CoA in this pathway can be catalyzed into butanol by aldol dehydrogenase. So, we inserted adhE2 to express aldehyde dehydrogenase. In addition, we enhanced the synthesis pathways of butyrate and butanol, by overexpressing thl and hbd, which encode two main enzymes, thiolysis enzyme and β-hydroxybutyryl-coenzyme, in the pathways, and by conjugating thiolysis enzyme (thl) and β-hydroxybutyryl-coenzyme (hbd) through a SpyTag/SpyCatcher system.
• 2) For the engineered E. coli, CALB gene was inserted to express CALB lipase from Candida antarcticum via a pet25b-T7-pelB-CALB-ChBD plasmid. By adding an affinity tag ChBD and pelB leader to CALB, we enhanced the reaction rate of CALB catalyzed esterification.

Project Goal

Our project aims to use shrimp shells from kitchen waste as raw materials to efficiently manufacture a novel bio-fuel butyl butyrate by an engineered Clostridium tyrobutyricum (C. tyrobutyricum). In this way, we hope to solve the current energy crisis and kitchen waste disposal problems simultaneously.

C. tyrobutyricum is chosen for our project for the following reasons:

• 1) It is able to secrete protease which aids in the degradation of shrimp shells, the nitrogen source for bacterial growth.
• 2) It is highly tolerant to butanol.
• 3) It contains a native synthesis pathway of butyrate, and a downstream intermediate butyryl-CoA can be catalyzed into butanol by Aldol dehydrogenase encoded by adhE2 gene [1] (Figure 1).

Butyrate and butanol can be synthesized into butyl butyrate by lipase.

Figure 1 Native synthesis pathway of butyrate and engineered synthesis pathway of butanol by adding adhE2 in C. tyrobutyricum

Missions of the project

In general, our project has three missions to be accomplished.

Mission 1: Constructing the synthesis pathway of butanol in C. tyrobutyricum

This mission was realized by inserting adhE2 gene from Clostridium acetobutylicum to a linearized pMTL-Pthl vector in C. tyrobutyricum using the Gibson assembly method.

In this way, we constructed a device part Pthl-RBS1-adhE2-Cpa fdx terminator (BBa_K4408008) to express edhE2 in C. tyrobutyricum. By adding edhE2 protein to catalyze the intermediate butyryl-CoA into butanol, we constructed a synthesis pathway of butanol in the bacteria. The detailed genetic circuit of the edhE2 expression system is shown in Figure 2.

Figure 2 Genetic circuit of pMTL-Pthl-adhE2

Mission 2: Optimizing the synthesis pathways of butyrate and butanol in C. tyrobutyricum

This mission was fulfilled by building two systems: a co-expression system and a SPY (SpyCatcher-SpyTag) system.

For the co-expression system, we constructed a pMTL-adhE2-thl-hbd recombinant plasmid on the basis of the pMTL-Pthl vector by Gibson assembly method. In addition to adhE2 gene, thl gene encoding thiolytic enzyme and hbd gene encoding β-hydroxybutyryl coenzyme from C. tyrobutyricum were also inserted into the plasmid. In this way, the three major enzymes in the synthesis pathways of butyrate and butanol are co-expressed in C. tyrobutyricum and their expressions are enhanced.

In this way, we constructed a device part Pthl-RBS1-adhE2-RBS1-thl-RBS1-hbd-Cpa fdx terminator (BBa_K4408009) to express edhE2, thl and hbd simultaneously in C. tyrobutyricum. The detailed genetic circuit of the edhE2, thl and hbd co-expression system is shown in Figure 3.

Figure 3 Genetic circuit of pMTL-Pthl-adhE2-thl-hbd

For the SPY (SpyCatcher-SpyTag) system, we constructed a pMTL-adhE2-SpyCatcher-thl-SpyTag-hbd recombinant plasmid. SpyTag and SpyCatcher are added into the plasmid to form SpyCatcher-thl and SpyTag-hbd fusion proteins. In this way, β-hydroxybutyryl coenzyme (hbd) and thiolytic enzyme (thl) merge together through the non-covalent interaction between SpyTag and SpyCatcher. By binding the two enzymes together, we can eliminate the accumulation of harmful intermediates, improve the transfer efficiency of intermediates, minimize metabolic crosstalk, enhance metabolic flow and improve product yield in the synthesis reactions of butyrate and butanol in C. tyrobutyricum.

In this way, we constructed a device part Pthl-RBS1-adhE2-RBS1-SpyCatcher-Linker 1-thl-RBS1-SpyTag-Linker 1-hbd-Cpa fdx terminator ( BBa_K4408010 ) to express edhE2, SpyCatcher-thl fusion protein and SpyTag-hbd fusion protein simultaneously in C. tyrobutyricum. The detailed genetic circuit of the edhE2, SpyCatcher-thl and SpyTag-hbd expression system is shown in Figure 4.

Figure 4 Genetic circuit of pMTL-Pthl-adhE2-SpyCatcher-thl-SpyTag-hbd and illustration of the SpyCatcher/SpyTag system

Mission 3: Engineering Escherichia coli (E. coli) to produce lipase

Lipase is needed to produce butyl butyrate from the two precursors, butyrate and butanol, manufactured by our engineered C. tyrobutyricum.

Lipase catalyzes the hydrolysis of oils and fats in nature. However, it can also accelerate reactions like esterification, transesterification, and alcoholysis reactions under suitable conditions [2]. Candida antarctica lipase B encoded by CALB has good catalytical function towards the synthesis of butyl butyrate. According to a recent study, CALB along with C. tyrobutyricum can reach a production of 34.7 g/L butyl butyrate from 80 g/L glucose and 10 g/L butanol [3]. ChBD is an affinity tag for chitin purification of proteins. ChBD-fusion CALB can facilitate the purification of CALB, thus enhancing the reaction rate of CALB catalyzed esterification.

Therefore we constructed a pet25b-T7-pelB-CALB-ChBD recombinant plasmid containing the CALB gene and ChBD gene and transformed it into E.coli. This engineered E.coli can produce lipase for the efficient production of our biofuel butyl butyrate.

In this way, we constructed a device part T7-RBS-pelB-CALB-Linker 2-ChBD-Cpa fdx terminator (BBa_K4408011) to express CALB-ChBD fusion protein in E.coli. The detailed genetic circuit of the expression system is shown in Figure 5.

Figure 5 Genetic circuit of pet25b-T7-pelB-CALB-ChBD