# Biohydrogen from Microalgae: Production and Applications
## 4 Genetic Engineering Approaches
### Targeted Mutagenesis  
On the other hand, targeted approaches turn out to be more advantageous and several proposals have been provided by genetic engineering tools to increase yields and overcome current limitations. As already mentioned, Fd receives electrons from the electron transport chain to address them to hydrogenase. Even under optimal conditions for enzyme expression, other assimilatory pathways compete for the reductants impairing the overall yield. Ferredoxin-NAD(P)\({}^{+}\) reductase (FNR) represents the main competitor shuttling electrons from Fd towards CO\({}_{2}\) fixation. To bypass this limitation, fusion protein has been designed. Among the most recent attempts, a fusion complex between Fd and hydrogenase in an in vivo culture of _Chlamydomonas reinhardtii_ was evaluated. The complex demonstrated higher production rates and greater oxygen tolerance than the sole enzyme [41]. Similar behavior was also observed in the _Chlamydomonas reinhardtii_ protein D1 mutant. A double amino acid substitution (L159I - N230Y) gave the mutant several new characteristics, including greater oxygen tolerance than the wild strain [42]. Similarly, _Chlamydomonas reinhardtii_ mutants knock out for flavodiiron protein (FDP) showed higher photoproduction of hydrogen than wild types. In this way, it is again demonstrated how by eliminating a competing pathway (for example the FDP-mediated O\({}_{2}\) photoreduction pathway), the electrons are preferentially conveyed towards the production of hydrogen. Furthermore, it has been seen that, even exposure to prolonged light pulses in these mutants do not direct the metabolism towards CO\({}_{2}\) fixation. It is, therefore, demonstrated how genetic engineering approaches together with actions on growth parameters are jointly useful to increase production [43]. LHC complexes exhibit a poor light energy conversion with more than 80% absorbed light energy wasted as fluorescence or heat and not addressed towards hydrogen production. Conversely, a truncated light-harvesting antenna has demonstrated improvements in terms of photoinhibition and light saturation phenomena. Afterward, the same approach has also shown encouraging results for hydrogen production: tla1 CC-4169 _Chlamydomonas reinhardtii_ mutant has exhibited to produce until six-time more hydrogen compared to the wild type strain with a light intensity of 350 \(\mu\)E m\({}^{-2}\) s\({}^{-1}\)[44; 45]. Non-coding RNA molecules, such as microRNA (miRNA) or long non-coding RNA (IncRNA), with regulatory function, are part of the most recent discoveries in microalgae and several studies have already exploited them for innovative approaches. In particular, miRNAs exhibit a regulatory function on the translation process by binding or degrading the messenger RNA and avoiding the corresponding protein synthesis. Transcriptomic studies showed that stressful situations in microalgae lead to an increase in these molecules which reflects the need to obtain immediate responses by the cell [46]. In _Chlamydomonas reinhardtii_, some endogenous miRNAs have overexpressed in S deprivation conditions. These observations led to the design of several artificial miRNAs (amiRNAs) to increase hydrogen yields by stimulating a faster oxygen consumption or repressing _psbA_ gene expression that encodes for PSII linked D1 protein [47; 48]. Similarly, optogenetic systems have also developed using properly blue light-inducible expression miRNAs. This gene control system has enhanced hydrogen production, confirming as a most promising tool [49]. Approaches related to genetic engineering require a fine upstream design and considerable resources. Certainly, specific approaches compared to random ones allow for better management of resources. Genome-scale metabolic reconstructions have already been used to direct choices in this sense for many species of prokaryotes and eukaryotes. For algal organisms, a similar tool has been developed using information from the literature.