The impact of fusion genes on the overexpression of enzymes for the heterologous production of β-phellandrene by Synechocystis mutants was investigated. The concept of overexpression of fusion genes was used in order to overcome the low expression level of these enzymes. Various constructs of the codon-optimized gene of β-phellandrene synthase (PHLS), along with the gene of geranyl diphosphate synthase (GPPS), were incorporated into the genomic DNA of Synechocystis sp. PCC 6803 following fusion with the highly expressed endogenous cpcB and cpcA genes, encoding the phycocyanin β- and α-subunits, respectively. Findings in this study indicated that the utilization of a strong promoter (cpc) in combination with the cpcB as a leader sequence was not by itself sufficient for cpcB.PHLS protein overexpression in the absence of the rest of the cpc operon genes (cpcA, cpcC2, cpcC1, cpcD). Significantly higher expression of the CpcB.PHLS fusion protein was achieved only when all cpc operon genes were present. In this case, the β-phellandrene yield was substantially greater compared with strains that also expressed the cpcB.PHLS fusion gene in the absence of the remainder cpc operon genes. Interestingly, when the cpcA was used in the leader sequence position, the CpcA.PHLS fusion protein caused the heterologous production of a mixture of terpenoid isomers, instead of β-phellandrene. This study extends previous findings in the field and provides new insights into the use of the fusion construct technology as a heterologous protein overexpression strategy for enzymes with slow catalytic activity.

研究了融合基因对Synechocystis突变体异源生产β-水芹烯的酶过表达的影响。为了克服这些酶的低表达水平，使用了融合基因的过表达概念。β-水芹烯合酶（PHLS）的密码子优化基因的各种构建体，以及香叶基二磷酸合酶（GPPS）的基因，被整合到Synechocystis sp。的基因组DNA中。与高度表达的内源性cpcB和cpcA融合后的PCC 6803基因，分别编码藻蓝蛋白的β-亚基和α-亚基。在这项研究中的发现表明，强的启动子（cpc）与cpcB作为前导序列的结合本身不足以使cpcB.PHLS蛋白在其余cpc操纵子基因（cpcA，cpcC2）缺失的情况下过表达。，cpcC1，cpcD）。仅当存在所有cpc操纵子基因时，CpcB.PHLS融合蛋白的表达才能显着提高。在这种情况下，与也表达cpcB.PHLS的菌株相比，β-芹菜烯的产量要高得多其余cpc操纵子基因不存在的情况下融合基因。有趣的是，当CPCA在前导序列位置被使用时，CpcA.PHLS融合蛋白引起的异源生产萜类化合物异构体的混合物的，而不是β水芹烯。这项研究扩展了该领域以前的发现，并为融合构建技术作为具有慢催化活性的酶的异源蛋白过表达策略的使用提供了新的见解。