Alternative Approaches for Enhancing Vitamin B6 Production
Previously, the assembly of enzymes to synthetic complexes, so- called "metabolons", revealed that flux imbalances could be prevented and spatial concentrations of the components of a heterologous pathway could be increased (Dueber et al., 2009; Moon et ah, 2010). This approach was successfully applied for enhancing increase of commercially attractive substances like mevalonate and glucaric acid (Dueber et ah, 2009; Moon et ah, 2010). Moreover, ALE of the host metabolic network to novel pathways through continuous selection for mutants with improved growth rates might be a powerful approach that can be applied to develop stable and efficient producers (Dragosits and Mattanovich, 2013). In combination with an inducible mutagenesis system, the ALE approach could accelerate the emergence of improved overproduction strains (Zhu et ah, 2015). In the evolved strains (1) the interactions between enzymes of the heterologous pathway with metabolic processes in the existing network, (2) the interference of metabolites in the existing network with the heterologous pathway, and (3) the loss of intermediates from the novel pathway by promiscuous activities of host enzymes might be minimized (Kim and Copley,
2012).
Low-level production of vitamin B6 might also be due to toxicity of the pathway intermediates and PLP (see above). PLP inhibits enzymes involved in DNA topology, DNA replication and translation. Therefore, it might interfere with essential cellular processes in the engineered production strains (Ohsawa and Gualerzi, 1981; Mizushina et al., 2003; Vermeersch et al., 2004). The efficient export of PN or PL to prevent their accumulation could be achieved by co-expression of a heterologous vitamin B6 biosynthetic pathway and PNP and PLP phosphatases together with (yet to be discovered) export systems for the vitamers. The overexpression of the native pdxP gene encoding the PNP phosphatase PdxP in S. meliloti indeed resulted an about 1.5-fold increase of vitamin B6 production (Tazoe et al., 2005; Nagahashi etal., 2008).
The efficiency of dephosphorylation and export of vitamin B6 could also be increased by co-evolution of mutually dependent organisms that cross-feed each other with a B6 vitamer and another metabolite, for which they are otherwise auxotrophic (Shong et al., 2012; Bernstein and Carlson, 2012; Groflkopf and Soyer, 2014; Jagmann and Philipp, 2014). Growth of the coculture over several passages might result in the emergence of spontaneous mutants showing increased growth due to enhanced vitamin secretion and/or production. Nowadays, genome re-sequencing is a cheap and straightforward methodology to identify the genotypes causing the phenotypes. The acquired beneficial mutations might be useful to generate strains with enhanced vitamin B6 production. The co-evolution approach could also result in the emergence of partially or completely novel vitamin B6 biosynthetic routes consisting of enzymes with improved promiscuous activities. Moreover, the toxicity of B6 vitamers might be reduced by strong overexpression of the potential PLP chaperone YggS from E. coli (YlmE in B. sub til is), which was shown to be involved in maintaining vitamin B6 homeostasis (Ito et al., 2013; Prunetti et al., 2016). However, the precise role of the conserved protein remains to be elucidated. Finally, the efficiency of the DXP-dependent vitamin B6 pathway could be improved by reducing the conversion of PNP to PLP and the selection of mutants that produce more PNP, and convert it to the biologically active vitamer through a promiscuous enzyme. Interestingly, it has been described that vitamin B6
auxotrophy of an E. coli pdxH mutant lacking the PNP oxidase is relieved by the acquisition of mutations in the pdxj gene (Man et al., 1996). A single amino acid exchange (Glyl94Ser) was sufficient to bypass the block in PLP synthesis (Fig. 1.2). Since the mutation did not fully restore growth, the genetic makeup seems to be suitable to enrich vitamin B6 overproducers through the acquisition of mutants that improve PNP synthesis. This approach could be pursued in combination with inhibitors that interfere with vitamin B6 metabolism (Pardini and Argoudelis, 1968).
Conclusions
As yet, attempts to engineer microbes for high-level production of vitamin B6 were unsuccessful. However, in the past years, the knowledge about vitamin B6 metabolism and its homeostasis has substantially increased. Moreover, several novel routes for vitamin B6 biosynthesis have been identified and characterized. These routes might serve as promising starting points for metabolic engineering, yielding in efficient overproducers. Furthermore, underground metabolism that is caused by promiscuous enzymes may be harnessed to generate novel routes for vitamin B6 biosynthesis. The rational engineering of microbes for vitamin B6 production might be still a powerful approach. For instance, to prevent the accumulation of toxic intermediates of heterologous vitamin B6 pathways, the enzymes production levels have to be tightly adjusted using available promoter libraries. Metabolic flux analyses are useful to identify and eliminate potential bottlenecks of heterologous pathways.
Acknowledgment
This work was supported by the Fonds der Chemischen Industrie (to FMC.), the Max-Buchner-Forschungsstiftung (MBFSSt 3381 to FMC), the Deutsche Forschungsgemeinschaft (DFG grants CO 1139/1-2 to FMC and GSC 226/2 to JR, respectively). We are grateful to Dr. Zoltan Pragai, Dr. Hans-Peter Hohmann for helpful comments and fruitful discussions.
