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.We successfully constructed 8 parts to engineer C. tyrobutyricum. We overexpressed several enzymes to enhance the synthesis pathway of butyrate and butanol. BBa_K4885000 and BBa_K4885002 were constructed to enhance the expression of deacetylase (Dac) to improve the acetylation and deacetylation interplay. BBa_K4885001, BBa_K4885005 and BBa_K4885006 were used to overexpress rate-limiting enzymes (bcd and crt). BBa_K4885003-BBa_K4885004 were used to overexpress CoA transferase (cat1) to inhibit the competing pathway of the byproduct, acetate. BBa_K4885012 was used to express adhE2 with a weaker promoter Ptkt to decrease butanol synthesis and indirectly increase butyrate yield to reach a better product ratio. In this way, we directly and indirectly reinforced the synthesis of butyrate and butanol in C. tyrobutyricum.
Table 1 Part list
| NO. | Name | Type | Description | Length |
|---|---|---|---|---|
| 1 | BBa_K4885000 | basic | Dac, NAD-dependent deacetylase | 723bp |
| 2 | BBa_K4885001 | basic | bcd, butyryl-CoA dehydrogenase coding sequence | 1140bp |
| 3 | BBa_K4885002 | composite | Pthl-adhE2-Dac | 3973bp |
| 4 | BBa_K4885003 | composite | Pcat1-Cat1 | 1720bp |
| 5 | BBa_K4885004 | composite | Pthl-adhE2-Pcat1-Cat1 | 4858bp |
| 6 | BBa_K4885005 | composite | Pthl-bcd-crt | 2593bp |
| 7 | BBa_K4885006 | composite | Pthl-adhE2-bcd-crt | 5112bp |
| 8 | BBa_K4885012 | composite | Ptkt-adhE2 | 2919bp |
The general design of our project is illustrated in Figure 1. In our project, we achieved the following five targets to construct a C. tyrobutyricum strain that produced butanol and butyrate with a favorable product ratio for the synthesis of butyl butyrate by lipase catalyzed esterification.
Figure 1 Flow chart of our project to synthesize butyl butyrate using microbial fermentation and CBD bound lipases
Target 1: Construction of a butanol synthesis pathway (dehydrogenase, adhE2)
2022 Worldshaper-NJBIOX team has engineered C. tyrobutyricum to express adhE2 and produce butanol. Inspired by their project, our team used the same part Pthl-adhE2 (BBa_K4408008) (plasmid pMTL-Pthl-adhE2) to express adhE2 in C. tyrobutyricum L319 to construct a butanol synthesis pathway (Figure 2). adhE2 gene was derived from Clostridium acetobutylicum ATCC 834. Pthl is a strong transcriptional promoter from C. tyrobutyricum L319. C. tyrobutyricum transfected with pMTL-Pthl-adhE2 plasmid was notated as Ct(adhE2).
Figure 2 Pthl-adhE2
Target 2: Direct enhancement of butyrate and butanol synthesis (deacetylase, Dac)
Build: Construction of pMTL-Pthl-adhE2-Dac plasmid
We constructed pMTL-Pthl-adhE2-Dac plasmid responsible for the expression of adhE2 and Dac with Pthl promotor in
C. tyrobutyricum L319.
Using the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 in the database as template, VAD-F and VDAC-R as primers, Vdac vector (8004 bp) was amplified by PCR. Using C. tyrobutyricum genome as template and DAC-F and DAC-R as primers, Dac gene fragment (767 bp) was amplified. Gibson assembly method was used to link Dac gene fragment to Vdac vector linearized vector. Colony PCR was performed on the transformed colonies (1050 bp) with DAC-PF and Pb-PR as primers. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, recombinant plasmid pMTL-Pthl-adhE2-Dac was obtained.
(a)Genetic circuit of pMTL-Pthl-adhE2-Dac;
(b) Verification of pMTL-Pthl-adhE2-Dac plasmid by colony PCR
Figure 3 Construction of pMTL-Pthl-adhE2-Dac plasmid
Test: Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-Dac
Ct(adhE2::Dac) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-Dac using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. Ct(adhE2) was used as the control.SDS-PAGE confirmed the overexpression of Dac protein (26 kDa) in Ct(adhE2::Dac) (Figure 4).
Growth performance analysis showed that overexpressing Dac in C. tyrobutyricum improved the growth of the strain, with the maximum OD600 increased by 33% (Figure 5).
HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2),
overexpressing Dac in C. tyrobutyricum improved the yield of butyrate (1.73 g/L) by 50% while did not change the butanol yield (5 g/L) (Figure 6-7), increasing the butyrate-to-butanol molar ratio from 0.19 to 0.29.
Butanol and butyrate synthesized by the fermentation of the engineered C. tyrobutyricum for 215 hours were used to synthesize butyl butyrate via CALB lipase catalyzed esterification reaction. Gas chromatograph was used to determine the yield. The yield of butyl butyrate in Ct(adhE2::Dac) increased 27% compared with that in Ct(adhE2) (Figure 8).
Figure 4 Verification of Dac protein overexpression in Ct(adhE2::Dac) by SDS-PAGE
Figure 5 Growth performance of Ct(adhE2::Dac)
Figure 6 Butyrate fermentation performance of Ct(adhE2::Dac)
Figure 7 Butanol fermentation performance of Ct(adhE2::Dac)
Figure 8 Production of butyl butyrate by Ct(adhE2::Dac)
Target 3: Inhibition of the competing pathway: acetate synthesis (CoA transferase, cat1)
Build: Construction of pMTL-Pthl-adhE2-Pcat1-cat1 plasmid
We constructed pMTL-Pthl-adhE2-Pcat1-cat1 recombinant plasmid to inhibit the synthesis of a by-product, acetic acid,
so that the synthesis of butanol and butyrate was enhanced indirectly.
Using the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 in the database as template and Vad-f and Vad-r as primers, the Vpthl-adhE2 vector (7985 bp) was amplified. Using C. tyrobutyricum genome as template and Cat-f and Cat-r as primers, cat1 gene fragment (1378 bp) was amplified. ,and Pcat-f and Pcat-r primers were used to amplify Pcat1 fragment. Gibson assembly method was used to link the cat1 gene fragment Pcat1 fragment to the Vpthl-adhE2 linearized vector. Colony PCR (1333 bp) was performed on the transformed colonies with primers cat-PF and Pb-PR. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, the recombinant plasmid pMTL-Pthl-adhE2-Pcat1-cat1 was obtained.
Figure 9 Genetic circuit of pMTL-Pthl-adhE2-Pcat1-cat1
Test: Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-Pcat1-cat1
Ct(adhE2::cat1) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-Pcat1-cat1 using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. Ct(adhE2) was used as the control.SDS-PAGE confirmed the overexpression of cat1 protein (47 kDa) in Ct(adhE2::cat1) (Figure10).
HPLC showed that after fermentation for 215 hours, compared with Ct(adhE2), overexpressing cat1 in C. tyrobutyricum improved the yield of butyrate by 130%, and decreased the yield of butanol by 26% (Figure 11), increasing the butyrate-to-butanol molar ratio from 0.3 to 1.1. In addition, the yield of acetate as a byproduct decreased significantly from 4 g/L to 1.2 g/L by overexpressing cat1.
Figure 10 Verification of cat1 protein overexpression in Ct(adhE2::cat1) by SDS-PAGE
Figure 11 Fermentation performance of butyrate, butanol and acetate of Ct(adhE2::cat1)
Target 4: Direct enhancement of butyrate and butanol synthesis pathway (rate-limiting enzymes, crt and bcd)
Build : Construction of pMTL-Pthl-adhE2-bcd-crt plasmid
Using the recombinant plasmid Pthl-adhE2 constructed by BBa_K4408008 in the database as template and VEAD-F and
veAD-r as primers, Vp-adhE2 vector (8001 bp) was amplified. Using C. tyrobutyricum genome as template, bcd gene
fragment (1196 bp) was amplified with bcd-f and BCD-R as primers, and crt gene fragment (813 bp) was amplified
with CRT-F and crt-R as primers. bcd gene fragment and crt gene fragment were fused into bcd-crt fragment by
fusion PCR. Gibson assembly method was used to link bcd-crt gene fragment to Vp-adhE2 linearized vector. Colony
PCR (1249 bp) was performed on the transformed colonies with primers bcd-PF and Pb-PR. The positive colonies
were transferred and the plasmid was extracted. After gene sequencing verification, the recombinant plasmid was
obtained: pMTL-Pthl-adhE2-bcd-crt.
Figure 12 Genetic circuit of pMTL-Pthl-adhE2-bcd-crt
Test : Construction of C. tyrobutyricum L319 with pMTL-Pthl-adhE2-bcd-crt
Ct(adhE2::bcd::crt) strain was obtained by conjugation of recombinant plasmid pMTL-Pthl-adhE2-bcd-crt, using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. Ct(adhE2) was used as the control.SDS-PAGE confirmed the overexpression of bcd and crt proteins in Ct(adhE2::bcd::crt) (Figure13).
The growth performance of Ct(adhE2::bcd::crt) was significantly better than Ct(adhE2), with the maximum OD600 reaching 10.2, an increase of 58% (Figure 14).
HPLC experiment showed that after fermentation for 215 hours, compared to the yields in Ct(adhE2), overexpressing bcd and crt increased the yield of butyrate by 20% and the yield of butanol by 40% (Figure 15).
Butanol and butyrate synthesized by the fermentation of the engineered C. tyrobutyricum for 215 hours were used to synthesize butyl butyrate via CALB lipase catalyzed esterification reaction. Gas chromatograph was used to determine the yield. 620 mg/L butyl butyrate was produced by Ct(adhE2::bcd::crt), which was 59% higher than the 390 mg/L yield by Ct(adhE2)(Figure 16).
Figure 13 Validation of the overexpression of bcd and crt in Ct(adhE2::bcd::crt) by SDS-PAGE
Figure 14 Growth performance of Ct(adhE2::bcd::crt)
Figure 15 Fermentation performance of butyrate, butanol and acetate of Ct(adhE2::bcd::crt)
Figure 16 Production of butyl butyrate by Ct(adhE2::bcd::crt)
Target 5: Use Ptkt promoter for adhE2 expression 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 this year. 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. adhE2 expression driven by Pthl in Ct(adhE2) showed too much exceeding synthesis of butanol to butyrate. So we replaced Pthl with the weaker promoter Ptkt to lower the production of butanol and increase the yield of butyrate in order to reach a more optimal butyrate-to-butanol product molar ratio (closer to 1:1) in our strain for the esterification of butyl butyrate.
Build : Construction of pMTL-Ptkt-adhE2 plasmid
Using pMTL-Pthl-adhE2 plasmid as the template and Vtkt-F and Vtkt-R as the primers, a linearized vector Vtkt was amplified (7825 bp). Using the C. tyrobutyricum genome as the template and TKT-F and TKT-R as the primers, a tkt fragment was amplified (300 bp). The linearized vector Vtkt and the tkt fragment were ligated by Gibson assembly. Colony PCR was performed on the transformed colonies (300 bp) with TKT-F and TKT-R as the primers. The positive colonies were transferred and plasmid was extracted. After gene sequencing verification, recombinant plasmid pMTL-Ptkt-adhE2 was obtained.
Figure 17 Genetic circuit of recombinant plasmid pMTL-Ptkt-adhE2
Figure 18 Colony PCR verification of pMTL-Ptkt-adhE2 recombinant plasmid
Test: Construction of C. tyrobutyricum L319 with pMTL-Ptkt-adhE2
Ct(Ptkt-adhE2) strain was obtained by conjugation of recombinant plasmid pMTL-Ptkt-adhE2 using E. coli CA434 as a donor strain and C. tyrobutyricum as a recipient strain. Ct(adhE2), also notated as Ct(Pthl-adhE2 in the figure, was used as the control.
HPLC showed that after fermentation for 215 hours (Figure 19), compared with Ct(Pthl-adhE2), Ct(Ptkt-adhE2) had increased the yield of butyrate by 66.5% and decreased the yield of butanol by 24.3%. Therefore, by using the weaker promoter Ptkt instead of Pthl, we raised the butyrate-to-butanol molar ratio from 0.29 to 0.63, much closer to the optimal ratio of 1:1. In addition, the production of acetate was reduced in Ct(Ptkt-adhE2) which was in accordance with the increased synthesis of butyrate.
Figure 19 Butyrate, butanol and acetate fermentation performance of Ct(Ptkt-adhE2)
