1 Introduction
Nature has assembled a multitude of microbial metabolic pathways constituting highly efficient reaction cascades that have been optimized during evolution [1]. In synthetic applications, cascade reactions allow for streamlined product formation via multiple reaction steps with the advantage to avoid the isolation of intermediates, thus saving resources, reagents, and time[2]. Although the balancing of enzyme ratiosin vivo is more complicated than in vitro , multi-step biocatalysis employing whole cells emerged as a powerful tool for the synthesis of value-added compounds [1, 3]. Precise and delicate fine-tuning of gene expression is required to balance individual enzyme amounts and activities for the construction of “designer cells” [2, 4]. Especially “artificial cascades” employing heterologous genes of diverse origin constitute a major challenge as they introduce novel enzymatic functions into the host [5]. On the one hand, it is crucial to provide sufficient enzyme amounts to sustain reasonable rates. On the other hand, too much overexpression, especially of more than one gene, can severely hamper host metabolism and interfere with its stability[6, 7]. A drain of resources from the central metabolism may affect overall enzyme resynthesis, cofactor supply, as well as enzyme folding and consequently cascade efficiency. Also, optimizations regarding the choice of the host strain, substrate uptake and flux can provide a systematic understanding of the in vivocascade [7-9]. A holistic approach comprising catalyst and reaction engineering allows controlling the product formation patterns [10].
Nowadays, plastics are ubiquitous in human life and cause severe litter problems. Thus, biodegradable polymers such as poly-caprolactone (PCL), polylactic acid, and polyhydroxyalkanoate have gained importance[11]. PCL can either be synthesized by the ring-opening polymerization of ε-caprolactone (ε-CL) or by the polycondensation of 6-hydroxyhexanoic acid (6-HA)[12]. Industrially, ε-CL is produced from cyclohexane through the Union Carbide Corporation (UCC) process, which suffers from serious environmental issues, a low cyclohexane conversion of 10-12 %, and only moderate selectivity of 85-90 %[13, 14]. Recently, two biocatalytic approaches to synthesize ε-CL from cyclohexane have been published. Pennec et al. demonstrated a one-pot reaction applying purified enzymes[15], whereas Karande et al. generated a whole-cell biocatalyst showing superior total turnover numbers[16]. Especially, the involvement of oxidoreductases constitutes a major challenge, as reasonable in vivo oxidoreductase activities depend on high expression levels of the active enzyme [17].
Karande et al. established a three-step cascade in P. taiwanensis VLB120 by introducing cytochrome P450 monooxygenase (Cyp), cyclohexanol dehydrogenase (CDH), and Baeyer-Villiger cyclohexanone monooxygenase (CHMO) genes from Acidovorax sp . CHX100 (Figure 1). Respective cascade development mainly focused on the monomer ε-CL and gave rise to a maximal activity of 22 U gCDW-1. Thereby, the first enzyme – the Cyp turned out to be rate-limiting (~20 U gCDW-1). The successive enzymes, the CDH, and the CHMO exhibited much higher activities of 80 U gCDW-1 and 170 U gCDW-1, respectively. In a separate study, the activity of cells containing only the Cyp could be more than doubled by expression system engineering [18]. A combination of this system with CDH and CHMO should guarantee high respective activities to prevent the accumulation of intermediates and, at the same time, keep the expression related metabolic burden reasonably low to allow for stable catalysis. Explicitly, oxygenases such as Cyps or Bayer-Villiger monooxygenases are prone to form reactive oxygen species via uncoupling reactions, which may hamper the catalytic prowess of the cells [7, 9]. Another point to be considered is that, due to the accumulation of the hydrolysis product 6HA, the approach of Karande et al . [16]suffered from restricted cascade selectivity. This study aimed at the optimization of this in vivo cascade by rational pathway engineering including the characterization of the involved enzymes as the basis for the rational assembly of the expression system. It is thereby crucial to balance enzyme activities without dissipating the cell’s resources.