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.