# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 2 Heterologous Production of 3-HP, a Frequent Intermediary from the AA Pathway
### Malonyl-CoA Route
coli_ was also overexpressed, and MCR fragments were codon-optimized. Later, [PERSON] et al. [41] focused on minimizing the imbalance previously verified in MCR-N and MCR-C expression [91]. To do that, MCR-C enzymatic activity was improved by directed evolution and the MCR-N expression level was decreased through the genome integration of 3 copies of the MCR-N gene [41]. Additionally, ACC from _E. coli_ was overexpressed and _pppR_ encoding an activator protein involved in the transcriptional regulation of the propionate catabolism operon was deleted, which blocked the production of the by-product propionate. In this study, 40.6 g/L of 3-HP was produced in fed-batch operation mode. To increase 3-HP concentrations and tolerance in _E. coli_, [PERSON] et al. [42] used the synthetic biology tool iterative CRISPR Enabled Trackable genome Engineering (iCREATE). This CRISPR-based tool allows the fast construction of modified genomes through multiplex and trackable editing and was used to test hundreds of thousands of genetic modifications in parallel. These modifications focused on the central carbon metabolism pathway to improve malonyl-CoA availability, 3-HP production from malonyl-CoA and pathways to increase strain tolerance to 3-HP. In addition to overexpressing MCR, they also overexpressed YdfG (3-hydroxy acid dehydrogenase from _E. coli_) to perform the second step of the pathway. RBS libraries were constructed in order to improve the expression of these genes and also AccADBC. To decrease by-product formation (lactate, acetate, formate and ethanol) and increase the metabolic flux toward malonyl-CoA, _ldhA_ (lactate dehydrogenase), _pflB_ (pyruvate formate-lyase), _adhE_ (alcohol dehydrogenase), _poxB_ (pyruvate oxidase), _ackA_ and _pta_ were deleted. Additionally, a temperature-sensitive mutation was introduced in _fabl_ (enoyl-acyl carrier protein) in order to decrease fatty acid biosynthesis at 37 \({}^{\circ}\)C and increase malonyl-CoA accumulation. Finally, several mutations were tested to increase 3-HP tolerance. Previously reported genes associated with low pH and organic acid tolerance, and regulators and transcription factors, were targeted. Mutations in oligopeptide ABC transporter periplasmic binding protein (OpPA), aldehyde dehydrogenase A (AldA), serine hydroxymethyltransferase (GlyA) and fused chorismate mutase/prephenate dehydatase (PheA) to increase their activity demonstrated to increase 3-HP tolerance and also production, with OppA mutation being the one that led to a higher 3-HP concentration. Overall, these modifications allowed constructing a strain that produced 60 times more 3-HP (30 g/L) than the control. Due to its potential, the heterologous production of 3-HP using the malonyl-CoA pathway has also been patented by academia [96] and industry [43] (Cargill). For example, [PERSON] et al. [43] implemented several modifications in addition to MCR expression, including ACC and _pmtAB_ overexpression to increase malonyl-CoA and NADPH availability, respectively; _ackA/pta/poxB_, _ldhA_/_mgSA_ (methylglyoxal synthase) and _pflB_ deletions to reduce acetate, lactate and formate accumulation, respectively; and _fabl_ mutation to indirectly increase malonyl-CoA accumulation. Concentrations of up 49 g/L of 3-HP in _E. coli_ were reported in this patent using these modifications [43]. As in _E. coli_, most studies related to 3-HP production in yeast (e.g., _S. cerevisiae_, _S. pombe_, _P. pastoris_) using the malonyl-CoA route use MCR and focus on increasing malonyl-CoA and cofactor supply [65]. In general, malonyl-CoA availability is improved by overexpressing the host ACC [92; 93; 94]. Other genes to increase acetyl-CoA supply may also be expressed, such as pyruvate decarboxylase (PDC1), acetyl-CoA synthase (ACS) and aldehyde dehydrogenase (ALD6) genes [92]. To improve NADPH availability, _GAPN_ or GAP dehydrogenase (_GAPDH_) genes, homologues of _gapA_, are frequently engineered [94; 92]. However, even when expressing multiple copies of these genes in the genome, the concentrations obtained in _S.