Enhancing microbial metabolite and enzyme production: current strategies and challenges

The metabolites and enzymes synthesized by microorganisms have been widely used as food (Mitsuhashi, 2014; Wendisch, 2014), pharmaceuticals (Elander, 2003; Endo, 2010), biofuels (Geddes et al., 2011), pesticides (Waldron et al., 2001; Yoon et al., 2004), and detergents (Shaligram and Singhal, 2010), as well as in the manufacturing process of these industrial products (Kirk et al., 2002; Merino and Cherry, 2007). They play important roles in our daily lives. The production methods used for useful metabolites and enzymes have improved since the time their importance was first established. If the genes involved in the synthesis of a metabolite or enzyme of interest are unknown, the production yield is enhanced by introducing random mutations into the chromosomes of the synthesizing microbe by ultraviolet (UV) irradiation or treatment with mutagens (Adrio and Demain, 2006). In addition, culture conditions have been adapted to further enhance production (Demain, 2000; Mukherjee et al., 2006). On the other hand, if the genes involved are known, their expression is also enhanced by metabolic engineering strategies such as gene disruption and overexpression using genetic modification techniques (Stephanopoulos et al., 1998; Adrio and Demain, 2010). When genetic modification of the producing microorganism is not possible because of difficulties in transformation, heterologous expression of the product of interest in other microbial species in which genetic modification can be more easily achieved has also been utilized for mass production (Stephanopoulos et al., 1998; Keasling, 2012). Primary metabolites essential for the normal growth of organisms are conserved between closely related microbial species, and their metabolic pathways including genetic components are almost fully elucidated. Therefore, metabolic engineering has been the chosen strategy used for increasing the microbial production of primary metabolites (Stafford and Stephanopoulos, 2001; Kern et al., 2007). About microbial enzymes, the coding genes are highly likely to be identified if both N-terminal amino acid sequences and molecular weights are not only identified by using highly purified samples but the genomic data of the producer microorganisms are also available. Searching a gene from the genomic data, on the basis of the N-terminal amino acid sequence and molecular weight, will help us identify an enzyme-coding gene. Once the gene has been identified, inducing overexpression of this gene in the original producer or another microbial host is one of the strategies adopted to increase the production of the enzyme (Demain and Vaishnav, 2009). Four strategies are considered to be effective in enhancing the production of primary metabolites. The first strategy is enhancing the expression of genes involved in metabolite synthesis. This strategy should be the most commonly used and reliable approach, but it does not always contribute to elevated production. In fact, we enhanced the expression of four enzyme genes, individually, that were involved in palmitic acid [C16fatty acid] synthesis, aiming to increase the production of free fatty acids (primary metabolites) in Aspergillus oryzae (Figure 1A-1). Overexpression of the fatty acid synthase (FAS) genes yielded a maximal increase in fatty acid production that was 2.8-fold more than that in the wild-type strain, whereas overexpression of the acetyl-CoA carboxylase (ACC) gene did not increase fatty acid production (Tamano et al., 2013). Overexpression of the two genes encoding ATP-citrate lyase (ACL) and palmitoyl-ACP thioesterase (TES) showed a moderate increase in fatty acid production (Tamano et al., 2013). Thus, each metabolic pathway is believed to consist of many sequential enzyme reactions, one of which has the lowest reaction rate and functions to regulate the rate of the whole pathway like a bottleneck. If an overexpressed gene encodes an enzyme that does not correspond to the bottleneck, there would be no resultant effect on production. In many cases, the bottleneck reactions are unknown; therefore, it is necessary to overexpress each gene involved in the synthesis of metabolites and determine which gene encodes for enzymes functioning at the bottleneck point of the pathway. Alternatively, one may simultaneously overexpress all genes involved in the synthesis in one cell; however, this is a difficult task to accomplish because it is time-consuming and labor-intensive to construct the mutant cell through DNA recombination technology. The second strategy by which primary metabolite production can be increased is the knockout of a reaction that degrades or converts the target metabolites. In Escherichia coli, a large amount of fatty acids was successfully produced