Metabolic engineering approach to elevate the carbon dioxide assimilation of cyanobacteria, which serves as a raw material for biofuel ethanol

蓝藻是一种快速生长的异养生物，需要辐射能，利用水作为电子供体。富含D-葡萄糖的蓝藻生物质可作为生物燃料乙醇的原料。尽管如此，通过CO_2浓缩机制从外部介质中积累的大量CO_2以无机形式存在于CO_2固定酶Rubisco周围。本研究旨在提高蓝藻生物质中无机碳向生物材料的转化率。为了提高蓝藻的碳利用率，采用代谢工程的方法来激活蓝藻的化学合成。氨甲酰磷酸合成酶（carbamoyloyl-phosphate synthetase，CPSase）是由谷氨酰胺和ATP合成氨甲酰磷酸的酶。CPSase由40 kDa的谷氨酰胺酶（OLN）亚基和120 kDa的合成酶（CPS）亚基组成。以粘粒cs 0499为模板，利用PCR技术从集胞藻PCC 6803中克隆到编码CPS亚基的carB基因。从集胞藻PCC 6803菌株中分离到含carB基因的质粒，转化大肠杆菌BL 21（DE 3）后，未检测到CPS亚基的活性。以大肠杆菌DH 5 α为模板，重复克隆carB基因。用细菌carB转化大肠杆菌C600表达CPS亚基。CPS亚基的比活力为146 mmol/g·min，在SDS-PHAGE上证实了CPS亚基的高产。并尝试将carB基因转化聚球藻7942-Spc。

Cyanobacteria are fast-growing photolithototrophs that require radiant energy, using water as electron donor. The biomass of cyanobacteria that are rich in D-glucose may serve as a raw material for biofuel ethanol. Despite this fact, large quantities of CO_2, accumulated from the external medium through a CO_2 concentrating mechanism, are in inorganic form around the CO_2 fixing enzyme Rubisco. The present study was undertaken to elevate the conversion of inorganic carbon to biomaterials of cyanobacterial biomass. To elevate the carbon utilization of cyanobacteria, a metabolic engineering approach to activate a chemosynthesis of cyanobacteria was performed. The enzyme of our concern was carbamoyl-phosphate synthetase (CPSase) that catalyzes the formation of carbamoyl phosphate from CO_2 and NH_3, derived from glutamine and ATP. CPSase consists of a 40kDa glutaminase (OLN) subunit and a 120kDa synthetase (CPS) subunit. The carB gene encoding CPS subunit was cloned from cyanobacterium Synechocystis sp. strain PCC6803 using PCR with a cosmid cs0499 as the template. Escherichia coli BL21(DE3) was transformed with the plasmid containing carB gene from Synechocystis sp. strain PCC6803, however, it represented no activity of CPS subunit. Cloning of carB gene was repeated using E.coli DH5α as the PCR template. E.coli C600 that was transformed with the bacterial carB expressed CPS subunit. Specific activity of CPS subunit was 146 mmol/g.min. High production of CPS subunit was confirmed on SDS-PHAGE. Attempt to transform Synechococcus sp. strain PCC7942-Spc with carB gene was also made.