# Trepenoid Metabolic Engineering in Photosynthetic Microorganisms
## 2 Pathway Modules in Terpenoid Biosynthesis
### General Considerations Spanning Across Nodes  
As discussed above, precise control of the heterologous biosynthetic pathways is often crucial to increase production. One way to help balance pathway flux across metabolic modules is to apply metabolite biosensors linked to feedback control mechanisms. Such a strategy can tie enzyme production (for example, terpene synthases) with internal metabolite pools (such as the enzyme's substrate or a key precursor molecule), which enables the production of the relevant enzyme exactly when required. Moreover, biosensors can be used to implement directed evolution methodologies, which connect compound concentration with growth or other responses. Two successful examples are the use of an NADPH sensor for neurosoporene production enhancement and an IPP sensor for lycopene production enhancement [58; 59]. [PERSON] and co-workers explored the notion that terpenoid production relies heavily on NADPH availability (and thus regeneration). They generated an NADPH fluorescent sensor to screen many _E. coli_ strain variants, selecting the ones with higher NADPH regeneration rates [58]. This allowed them to improve 25-fold the production titers of the carotenoid neurosoporene. [PERSON] and [PERSON] constructed an elaborate IPP-controlled genetic circuit: low IPP causes high mutagenesis rate while, as the IPP concentration increases, the mutation rate drops [59]. Since this is tied to a fluorescent reporter, the researchers could select the strains with lower fluorescence (which had higher IPP production rates) and confirm that these strains could accumulate significantly more lycopene.  
Flux reporters such as the carotenoids lycopene and beta-carotene are commonly used as indicators of the terpenoid yield since they give a colored phenotype that can be assessed by absorption or fluorescence. This has been exploited in studies to identify elements such as transcription factors that have an effect on production, coupled with directed evolution or high throughput engineering approaches [60; 61; 62]. There are, however, some problems with using lycopene as a flux reporter in that changes in the cellular redox status result in oxidation of lycopene and loss of color--thereby interfering with its effectiveness as a pathway flux reporter [62]. In addition, accumulation is affected by prenyl phosphate metabolism influencers and may not relate directly to a core isoprenoid (MEP/MVA) pathway flux. These considerations can render lycopene a poor reporter of pathway flux in some cases. Alternative reporters such as isoprene may be more useful, but assays are not as high throughput or as facile as the colored products.  
It should be noted that MEP and MVA pathways both consume reducing power by using NADPH and NADH, respectively. The availability of these redox partners as well as haem molecules (serving as enzyme cofactors) and the precursors acetyl-coA (for the MVA pathway) and pyruvate and glyceraldehyde 3-phosphate (of the MEP pathway) are limiting steps. Therefore, the introduction of pathways that enable the recycling of cofactors or increase their availability have been used successfully to increase terpenoid production [63; 64; 65; 66; 67].  
Often, it is not easy to optimize all four nodes and produce a terpenoid of interest in a single organism. The use of dual-organism production systems is an interesting alternative. An example has been implemented in the production of oxygenated products of taxadiene. _E. coli_ was optimized to produce taxadiene, which was oxygenated by a yeast expressing taxadiene 5\(\alpha\)-hydroxylase (a cytochrome P450) and the P450 reductase [68]. This microbial consortium approach shows that different organisms are needed to optimally catalyze different reactions where the enzymes require bespoken environments provided by different hosts.