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Abstract

Our project aims to use shrimp shells from kitchen waste as raw materials to efficiently manufacture a biofuel butyl butyrate via biosynthetic process. Butyl butyrate can be synthesized by lipase using butyrate and butanol. We engineered Clostridium tyrobutyricum (C. tyrobutyricum) to express adhE2 and overexpress thl and hbd, so that it can produce both butyrate and butanol with high yields. We also engineered E. coli to produce Candida antarctica lipase B (CALB) which can catalyze the synthesis of butyl butyrate from butyrate and butanol. With laboratory work, we proved that the engineered C. tyrobutyricum could grow actively with high yields of both butanol and butyrate, and exogenous lipase CALB produced by the engineered E. coli had good enzyme activity in synthesizing butyl butyrate.

Overview of the project

Our project aims to use shrimp shells from kitchen waste as raw materials to efficiently manufacture a biofuel butyl butyrate via biosynthetic process. Butyl butyrate can be synthesized by lipase using butyrate and butanol. Native Clostridium tyrobutyricum (C. tyrobutyricum) can only synthesize butyrate. We engineered Clostridium tyrobutyricum (C. tyrobutyricum) to express adhE2 so that it can synthesize butanol as well. We further engineered C. tyrobutyricum to overexpress thl and hbd, so that it can produce both butyrate and butanol with high yields. We also engineered E. coli to produce Candida antarctica lipase B (CALB) which can catalyze the synthesis of butyl butyrate from butyrate and butanol.

As for whether our engineered bacteria can achieve the desired effect, the following three aspects need to be proved:

How did we carry out the tests?

Experiments

(1) Cell samples

Cell samples used for our experiments included the following:

Control: wild type C. tyrobutyricum.

Ct(adhE2::thl::hbd): C. tyrobutyricum conjugatively transformed with pMTL-Pthl-adhE2-thl-hbd using E. coli CA434 as the donor strain, co-expressing adhE2, thl and hbd.

Ct(adhE2): C. tyrobutyricum conjugatively transformed with pMTL-Pthl-adhE2 using E. coli CA434 as the donor strain, expressing adhE2.

Engineered E. coli: E. coli transformed with pet25b-T7-pelB-CALB-ChBD plasmid, expressing CALB-ChBD fusion protein.

(2) Cell culturing

The culture medium used for cultivating E. coli was Luria Bertani (LB) medium, which consisted of 1% peptone, 1% sodium chloride, 0.5% yeast powder, and the fermentation medium for E. coli consisted of 1.75% mannitol and 2.25% peptone. The enriched medium for cultivating C. tyrobutyricum consisted of 1% peptone, 1% beef extract, 0.5% sodium chloride, 0.5% glucose, 0.3% anhydrous sodium acetate, 0.3% yeast powder, 0.1% soluble starch, and 0.05% cysteine hydrochloride (pH 6.5). The fermentation medium TGY of C. tyrobutyricum was shrimp shell powder medium, which consisted of 2.5%-3% shrimp shell powder, 0.5% sodium chloride peptone, 0.15% potassium hydrogen phosphate, 0.06% magnesium sulfate heptahydrate, 0.003% ferrous sulfate heptahydrate, 2% glucose, 1% yeast powder, and 0.13% cysteine hydrochloride, pH 6.0. E. coli was cultured at 37℃ 200 rpm in a triangular flask, induced fermentation at 20℃ 200 rpm in a triangular flask, and C. tyrobutyricum was cultured in an anaerobic flask at 37℃.

(3) Methods

Growth performance analysis:

Ultraviolet spectrophotometer was used to measure OD600 value to evaluated the growth of the cells.

Product extraction and yield analysis:

Hexadecane was used as an extractant of butyric acid, butanol and butyl butyrate. HPLC was used to determine the concentration of butyric acid, butanol and butyl butyrate. We added 50μl /100mL immobilized CALB enzyme to the engineered C. tyrobutyricum to synthesize butyl butyrate, and HPLC was used to determine the yield of butyl butyrate.

Protein gel electrophoresis:

we took the bacterial solution freshly cultured to OD600~2.0, centrifugated it with 10000×g for 1 min to remove the supernatant, and then resuspended it with 1mL PBS buffer, pH 7.0, centrifugated it with 10000×g for 1 min to remove the supernatant, and then added 0.5mL PBS buffer for resuspension. The cells were broken by ultrasound for 10 min, and the broken transparent protein solution was centrifuged for 5 min at 10000×g to remove the residual cell fragments and insoluble substances. Then we took the supernatant 160μl，added 4×SDS protein electrophoresis buffer solution and heated it at 100℃ for 10 min. After it was cooled to room temperature, sampled it for protein electrophoresis.

Determination of enzyme activity of lipase:

butyric acid and butanol (5 g/L each) were used as substrates, and hexadecane was used as extractant through two-phase catalysis. Butyl butyrate generated in unit time was measured by gas phase to determine the activity of lipase. 1 U (μmol/min) is defined as the amount of the enzyme that catalyzes the conversion of one μmol of substrate per minute.

Immobilization of lipase:

20 mL of free lipase with chitin binding domain (CALB-ChBD fusion protein) was mixed with 2 g of chitin pellets, and fully adsorbed for 2 h at 20℃ 100 rpm in a shaker to obtain chitin lipase pellets.

Results

(1) Growth of the engineered C. tyrobutyricum

The growth test showed that the growth performance of the engineered strain was better than the wild type (control), with the maximum OD600 increased by 21% (Figure 1).

Figure 1. Growth of the engineered and wild type C. tyrobutyricum

Ct(adhE2::thl::hbd): C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd;

Control: wild type C. tyrobutyricum

(2) Butanol and butyl acid production of the engineered C. tyrobutyricum

HPLC experiment showed that the engineered C. tyrobutyricum overexpressing adhE2, thl and hbd could produce butanol. The experiment also showed that the yields of butyrate and butanol were increased by 30% and 33%, respectively, in this strain, compared to the strain without thl and hbd overexpression (Figure 2).

Since they are the precursors for butyl butyrate synthesis, the final butyl butyrate production was up to 590 mg/L in this strain, an increase by 1.1 fold compared to the strain without thl and hbd overexpression (Figure 3).

Figure 2 Yields of butyrate and butanol of C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd (adhE2::thl::hbd) vs. C. tyrobutyricum transformed with pMTL-Pthl-adhE2 (adhE2)

Figure 3 Butyl butyrate yield from C. tyrobutyricum transformed with pMTL-Pthl-adhE2-thl-hbd (adhE2::thl::hbd) vs. that from C. tyrobutyricum transformed with pMTL-Pthl-adhE2 (adhE2)

(3) Enzyme activity of CALB produced by the engineered E. coli

Protein gel electrophoresis showed that the engineered E. coli produced Candida antarctica Lipase B (CALB) (Figure 4).

In the enzyme activity assay, the exogenously expressed lipase was found to have good activity, showing the highest enzyme activity of 110 U/mL at 120 min (Figure 5). 220 mg/L butyl butyrate was obtained by catalyzing C. tyrobutyricum transformed with pMTL-Pthl-adhE2. After immobilization by chitin pellets, the enzymatic activity of CALB increased by 24% to 136 U/mL, and the final catalytic butyl butyrate production was increased by 28% to 280 mg/L.