## 4 Renewable Carbon Sources for the Biosynthesis of 3-HP
### Microbial Production of 3-HP from Glycerol
#### 4.2.2 Biosynthesis of 3-HP via the Coenzyme A-Independent Pathway  
The biosynthesis of 3-HP from glycerol via the coenzyme A-independent pathway has been extensively studied. However, it is important to note that imbalances in the activity of key enzymes of the coenzyme A-independent pathway usually lead to the accumulation of the toxic intermediate 3-HPA. Other factors limiting the production of 3-HP from glycerol include the unbalanced growth-production relationship [84] and the availability of coenzyme B\({}_{12}\). Finally, the conversion of glycerol to 3-HP is accompanied by a net accumulation of reducing equivalents, and it is essential to achieve intracellular redox balance for microorganisms to efficiently metabolize glycerol for the synthesis of 3-HP [85]. Researchers have attempted to enhance the activity of ALDH and balance the production pathway to improve the 3-HP titer obtained by the coenzyme A-independent pathway. A novel aldehyde dehydrogenase (GabD4) from _Cupriavidus necator_ was found to have high catalytic activity, and it was modified by site-saturation mutagenesis based on homology modeling. Finally, an engineered _E. coli_ expressing a GabD4 mutant with 40% higher enzyme activity produced 71.9 g/L of 3-HP in fed-batch fermentation [32]. To enhance the expression of ALDH, the T7 expression system was introduced into _K. pneumoniae_ and the 3-HP yield of the recombinant strain was enhanced by 3.24-fold [86]. [PERSON] et al. [27] used the tac promoter with three tandem repeats to initiate the transcription of _punC_ (encoding ALDH in _K. pneumoniae_), and the engineered _K. pneumoniae_ reached a titer of 102.61 g/L in fed-batch fermentation. However, it is impossible to fully balance the activity of ALDH and GDHt by increasing ALDH activity alone. Regulatory cassettes containing promoters of different strength and double ribosome binding sites were used to express ALDH and GDHt, which showed that 3-HPA accumulation could be completely eliminated when the expression level of ALDH was approximately eight-fold higher than that of GDHt [87]. In a different approach, the catalytic activity of GDHt was precisely downregulated by 5 UTRs engineering, and the yield reached 0.97 g of 3-HP/g glycerol in fed-batch fermentation, which is the highest yield for the production of 3-HP from glycerol reported to date [88]. The main factors limiting the conversion of glycerol to 3-HP are unsuitable glycerol assimilation rates together with unbalanced production and growth relationships. _K. pneumoniae_ possesses a thoroughly investigated glycerol metabolic pathway that can rapidly metabolize glycerol. The carbon flux from glycerol towards 3-HP production rather than growth in _K. pneumoniae_ has been achieved by decreasing the rate of glycerol assimilation through the deletion of glycerol kinase (GlpK) [89; 90]. A tryptophan operon-assisted CRISPR interference (CRISPRi) system that switches the glycerol oxidation and reduction pathways in _K. pneumoniae_ has been reported, and the CRISPR-engineered strain produced 88.8 g/L of 3-HP in fed-batch fermentation [28]. Compared to _K. pneumoniae_, glycerol is utilized less efficiently by _E. coli_. Researchers attempted to knock out _glpK_ in _E. coli_ to redirect the metabolic flux towards 3-HP production. However, the production of 3-HP did not improve [91]. Interestingly, a significant increase in 3-HP production (from 28.1 g/L to 40.5 g/L) was achieved by overexpressing GlpK and knocking down GlpR (glycerol pathway blocker) to promote glycerol assimilation in _E. coli_[84]. It is necessary to overexpress glycerol dehydratase reactivase (GdrAB) to reactivate coenzyme B\({}_{12}\) for some chassis that cannot synthesize coenzyme B\({}_{12}\) naturally or produce 3-HP via the glycerol pathway under aerobic conditions. A report showed that oxygen can induce the formation of inactivated coenzyme B\({}_{12}\), which further causes the inactivation of GDHt by binding tightly to it, resulting in limited flux from glycerol to 3-HP [54]. [PERSON] et al.