Microbial Chemistry, Department of Chemistry-Ångström Laboratory, Uppsala University, Box 523, 75120, Uppsala, Sweden.
Microb Cell Fact. 2022 Feb 1;21(1):17. doi: 10.1186/s12934-022-01738-z.
Cyanobacteria, photosynthetic microorganisms, are promising green cell factories for chemical production, including biofuels. Isobutanol, a four-carbon alcohol, is considered as a superior candidate as a biofuel for its high energy density with suitable chemical and physical characteristics. The unicellular cyanobacterium Synechocystis PCC 6803 has been successfully engineered for photosynthetic isobutanol production from CO and solar energy in a direct process.
Heterologous expression of α-ketoisovalerate decarboxylase (Kivd) is sufficient for isobutanol synthesis via the 2-keto acid pathway in Synechocystis. With additional expression of acetolactate synthase (AlsS), acetohydroxy-acid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), and alcohol dehydrogenase (Slr1192), the Synechocystis strain HX42, with a functional 2-keto acid pathway, showed enhanced isobutanol production reaching 98 mg L in short-term screening experiments. Through modulating kivd copy numbers as well as the composition of the 5'-region, a final Synechocystis strain HX47 with three copies of kivd showed a significantly improved isobutanol production of 144 mg L, an 177% increase compared to the previously reported best producing strain under identical conditions.
This work demonstrates the feasibility to express heterologous genes with a combination of self-replicating plasmid-based system and genome-based system in Synechocystis cells. Obtained isobutanol-producing Synechocystis strains form the base for further investigation of continuous, long-term-photosynthetic isobutanol production from solar energy and carbon dioxide.
蓝藻是一种光合微生物，是化学产物（包括生物燃料）生产的有前途的绿色细胞工厂。异丁醇是一种四碳醇，因其具有较高的能量密度和适宜的化学物理特性，被认为是一种较好的生物燃料候选物。单细胞蓝藻集胞藻 PCC 6803 已被成功设计用于通过直接过程从 CO 和太阳能中生产光合异丁醇。
α-酮异戊酸脱羧酶（Kivd）的异源表达足以通过 2-酮酸途径在集胞藻中合成异丁醇。通过额外表达乙酰乳酸合酶（AlsS）、乙酰羟酸异构还原酶（IlvC）、二羟酸脱水酶（IlvD）和醇脱氢酶（Slr1192），具有功能性 2-酮酸途径的集胞藻 HX42 菌株在短期筛选实验中显示出增强的异丁醇生产能力，达到 98mg/L。通过调节 kivd 拷贝数以及 5'-区的组成，最终的集胞藻 HX47 菌株携带三个 kivd 拷贝，其异丁醇产量显著提高到 144mg/L，与相同条件下先前报道的最佳生产菌株相比，提高了 177%。
这项工作证明了在集胞藻细胞中使用自我复制质粒系统和基因组系统组合表达异源基因的可行性。获得的产异丁醇集胞藻菌株为进一步研究从太阳能和二氧化碳连续、长期光合生产异丁醇奠定了基础。
