Ethanol synthesis by genetic engineering in cyanobacteria

Abstract

Cyanobacteria are autotrophic prokaryotes which carry out oxygenic photosynthesis and accumulate glycogen as the major form of stored carbon. In this research, we introduced new genes into a cyanobacterium in order to create a novel pathway for fixed carbon utilization which results in the synthesis of ethanol. The coding sequences of pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh) from the bacterium Zymomonas mobilis were cloned into the shuttle vector pCB4 and then used to transform the cyanobacterium Synechococcus sp. strain PCC 7942. Under control of the promoter from the rbcLS operon encoding the cyanobacterial ribulose-1, 5-bisphosphate carboxylase/oxygenase, the pdc and adh genes were expressed at high levels, as demonstrated by Western blotting and enzyme activity analyses. The transformed cyanobacterium synthesized ethanol, which diffused from the cells into the culture medium. As cyanobacteria have simple growth requirements and use light, CO2, and inorganic elements efficiently, production of ethanol by cyanobacteria is a potential system for bioconversion of solar energy and CO2 into a valuable resource.

FIG. 1

Plasmid vectors for expressing Z. mobilis PDC (pdc) and ADH II (adh) in cyanobacteria. All of the plasmids were constructed in the shuttle vector pCB4 for transforming Synechococcus sp. strain PCC 7942. In pCB4-Rpa, the pdc and adh genes are under the control of the promoter of the rbcLS operon (labelled R). In both pCB4-LRpa and pCB4-LR(TF)pa, pdc expression and adh expression are driven by a combination of the rbcLS promoter and the E. coli lac promoter (labelled L). The ribosome-binding site and the start codon of the rbcL gene were fused in frame to the second codon of the pdc gene in pCB4-LR(TF)pa. The arrows indicate the directions of transcription and translation. The position of the effective translation initiation codon (ATG) for the pdc and adh genes is indicated. The transcription terminator sequence of the adh gene is represented by a solid box (labelled T). The restriction sites used in cloning are shown (B, BamHI; P, PvuII; E, EcoRI, S, SalI; X, XbaI; Xh, XhoI). Letters in parentheses indicate restriction sites which were eliminated by blunt end ligation.

FIG. 2

Northern blot analysis of cyanobacterial RNA. Samples (8 μg) of total RNA isolated from wild-type Synechococcus sp. strain PCC 7942 (lanes 1) and cyanobacterial cells transformed with the ethanol synthesis construct pCB4-LRpa (lanes 2) were fractionated by denaturing agarose gel electrophoresis and blotted onto a nitrocellulose membrane. The membrane was hybridized with the pdc probe to detect the dicistronic transcript pdc-adh and was hybridized with the sodB probe to check the overall quality of the RNA preparations. The positions of RNA molecular size markers (in kilobases) are indicated on the left. The arrows indicate the positions of the expected cotranscription products obtained from the pdc and adh genes (3.2 kb) and the sodB transcript (0.7 kb).

FIG. 3

Western blot analysis of cyanobacterial proteins. Lanes 2 through 5 contained 8-μg portions of proteins extracted from the cyanobacterial cells harboring the shuttle vector pCB4 (lane 2) and the following vectors containing the pdc and adh genes: pCB4-Rpa (lane 3), pCB4-LRpa (lane 4), and pCB4-LR(TF)pa (lane 5). Proteins extracted from E. coli harboring pLOI295 containing the pdc and adh genes were used as a positive control (lane 1). The blots were probed with antiserum directed against Z. mobilis PDC protein and antiserum raised against Z. mobilis ADH II protein.

FIG. 4

Cell growth and ethanol synthesis in Synechococcus sp. strain PCC 7942 transformed with pCB4-LRpa. Cells were grown at 30°C in the presence of light in a 500-ml liquid batch culture aerated by forcing air through a Pasteur pipette. Samples were taken at intervals in order to monitor cell growth (OD₇₃₀) and ethanol accumulation in the culture medium. The PDC and ADH activities in cell lysates on day 5 were 320 and 170 nmol · min⁻¹ · mg of total protein⁻¹, respectively. The values are the means of two or three values obtained with different samples.

FIG. 5

Photosynthesis and photoassimilate metabolism in cyanobacteria. Abbreviations: 2-PGA, 2-phosphoglyceric acid; 3-PGA, 3-phosphoglyceric acid; F6P, fructose-6-phosphate; PEP, phosphoenolpyruvic acid; RuBP: ribulose 1,5-bisphosphate; TCA cycle, tricarboxylic acid cycle; acetyl CoA, acetyl coenzyme A. The pathway at the upper right is the added pathway for ethanol synthesis.