Helmholtz-Centre for Environmental Research - UFZ, Permoserstr, Leipzig, Germany.
Plant Biotechnol J. 2023 Oct;21(10):2074-2083. doi: 10.1111/pbi.14113. Epub 2023 Jul 13.
The photosynthetic light reaction in cyanobacteria constitutes a highly attractive tool for productive biocatalysis, as it can provide redox reactions with high-energy reduction equivalents using sunlight and water as sources of energy and electrons, respectively. Here, we describe the first artificial light-driven redox cascade in Synechocystis sp. PCC 6803 to convert cyclohexanone to the polymer building block 6-hydroxyhexanoic acid (6-HA). Co-expression of a Baeyer-Villiger monooxygenase (BVMO) and a lactonase, both from Acidovorax sp. CHX100, enabled this two-step conversion with an activity of up to 63.1 ± 1.0 U/g without accumulating inhibitory ε-caprolactone. Thereby, one of the key limitations of biocatalytic reactions, that is, reactant inhibition or toxicity, was overcome. In 2 L stirred-tank-photobioreactors, the process could be stabilized for 48 h, forming 23.50 ± 0.84 mm (3.11 ± 0.12 g/L) 6-HA. The high specificity enabling a product yield (Y ) of 0.96 ± 0.01 mol/mol and the remarkable biocatalyst-related yield of 3.71 ± 0.21 g /g illustrate the potential of producing this non-toxic product in a synthetic cascade. The fine-tuning of the energy burden on the catalyst was found to be crucial, which indicates a limitation by the metabolic capacity of the cells possibly being compromised by biocatalysis-related reductant withdrawal. Intriguingly, energy balancing revealed that the biotransformation could tap surplus electrons derived from the photosynthetic light reaction and thereby relieve photosynthetic sink limitation. This study shows the feasibility of light-driven biocatalytic cascade operation in cyanobacteria and highlights respective metabolic limitations and engineering targets to unleash the full potential of photosynthesis.
蓝藻的光合作用反应是一种极具吸引力的生产性生物催化工具，因为它可以利用阳光和水分别作为能源和电子的来源，为氧化还原反应提供高能还原当量。在这里，我们描述了在集胞藻 PCC 6803 中首次进行的人工光驱动氧化还原级联反应，以将环己酮转化为聚合物构建块 6-羟基己酸（6-HA）。共表达来自 Acidovorax sp. CHX100 的 Baeyer-Villiger 单加氧酶（BVMO）和内酯酶，使两步转化的活性高达 63.1±1.0 U/g，而不会积累抑制性 ε-己内酯。由此，克服了生物催化反应的一个关键限制因素，即反应物抑制或毒性。在 2 L 搅拌式光生物反应器中，可以稳定该过程 48 h，形成 23.50±0.84 mm（3.11±0.12 g/L）6-HA。高特异性使产物收率（Y）达到 0.96±0.01 mol/mol，生物催化剂相关收率达到 3.71±0.21 g/g，这表明在合成级联中生产这种无毒产物具有潜力。发现对催化剂的能量负担的精细调整至关重要，这表明可能由于与生物催化相关的还原剂去除而使细胞的代谢能力受到限制。有趣的是，能量平衡表明生物转化可以利用光合作用光反应产生的多余电子，并由此缓解光合作用的汇限制。本研究表明在蓝藻中进行光驱动生物催化级联操作的可行性，并强调了各自的代谢限制和工程目标，以释放光合作用的全部潜力。
