The previously constructed strain E. coli BOX3.3 ∆4  was used as a core for evaluating the efficiency of the functional reversal of FAO upon the action of various native acyl-CoA dehydrogenases  (Table 2). In this strain, the ackA, pta, poxB, ldhA, and adhE genes encoding enzymes of the mixed-acid fermentation pathways involving pyruvic acid and its direct derivative, acetyl-CoA, a key metabolite precursor in the reactions of inverted FAO, were inactivated. In addition, the expression of the atoB and fadB genes encoding the FAO enzymes, which ensure the formation of 3-hydroxybutyryl-CoA from acetyl-CoA, was enhanced in the strain, as well as the expression of the thioesterase II gene, tesB, which can serve as a terminating enzyme that ensures the formation of carboxylic acids from acyl-CoA intermediates of FAO. In the strain, the nonspecific thioesterase YciA gene was inactivated in order to further reduce the competitive conversion of acetyl-CoA to acetic acid, and the main acyl-CoA dehydrogenase gene, fadE, was inactivated in order to prevent multiple reversal of FAO. As a result, during test-tube fermentation, the strain was capable of synthesizing 3-hydroxybutyric acid from glucose up to ~6 mM under microaerobic and 3-hydroxybutyric acid up to ~4 mM under anaerobic conditions resulting from partial one-turn inversion of FAO .
Reversed FAO reactions catalyzed by 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA dehydrogenase are NADH-consuming. Thus, anaerobic conditions that exclude intense oxidation of reduced equivalents in the respiratory electron transport chain with the participation of oxygen as a terminal electron acceptor are more preferable to support multiple cycle reversal. Therefore, the efficiency of FAO reversal in the studied strains was evaluated by the concentrations of marker compounds secreted by recombinants in the anaerobic stage of two-phase aerobic–anaerobic fermentation. The corresponding cultivation process, including an aerobic stage of biomass accumulation followed by an anaerobic productive stage, was chosen because E. coli strains deficient in mixed acid fermentation pathways are not capable of anaerobic growth , but retain metabolic activity in the absence of aeration.
During anaerobic utilization of glucose, core strain BOX3.3 ∆4 synthesized acetic and lactic acids, as well as ethanol (Table 3) as the main products of the carbon substrate consumption, without secreting detectable amounts of products of complete and moreover, repeated reversal of FAO due to the deletion of the fadE gene (Table 4).
It should be noted that the strain did not secrete acyl-CoA derived carboxylic acids, despite the presence of intact fabI and ydiO genes in the chromosome. This indicated that the native levels of expression of the corresponding genes could not provide sufficient acyl-CoA dehydrogenase activity in the cell for successful functional reversal of FAO. Indeed, in the case of FabI, acyl-CoA dehydrogenase activity is secondary to the main enoyl-ACP reductase activity of this protein , and to ensure efficient reduction of crotonyl-CoA, which is required for FAO reversal in recombinant strains, it is necessary to increase the expression of the fabI gene . On the other hand, the expression of the ydiO gene encoding the acyl-CoA dehydrogenase of anaerobic FAO  can be repressed in the presence of oxygen . The aerobic conditions used for the accumulation of the biomass of the BOX3.3 ∆4 strain prevented the formation of such a level of the corresponding protein in the cells during the growth stage that would be sufficient for its effective action in the subsequent biosynthetic stage. In addition, in the absence of fatty acids in the medium, the expression of fad regulon genes, including fadE, is repressed in E. coli by the transcriptional regulator FadR, and the activity of the corresponding enzymes in the cell is severely limited . Taken together, this indicated that in order to ensure the activities of acyl-CoA dehydrogenases in the recombinants, which are necessary for the efficient reversal of FAO, an increase in the expression of the corresponding genes was an essential condition.
In the BOX3.3 ∆4 strain, the expression of the genes for acetyl-CoA-C-acetyltransferase, atoB, and thioesterase II, tesB, was controlled by the lambda phage PL promoter, which is one of the “strongest” for E. coli, while the Ptrc-ideal-4 promoter, somewhat inferior to PL promoter, was located upstream from the 3‑hydroxyacyl-CoA dehydrogenase gene, fadB . The corresponding promoters in the artificial regulatory elements containing an effective ribosome-binding site of φ10 gene from the T7 phage were located before the indicated genes to ensure, first of all, the possibility of efficient initiation of FAO reversal and the subsequent formation of detectable products from the CoA intermediates of the cycle, while the intensity of the intermediate reactions could be somewhat decreased. Thus, in order to prevent a potential imbalance between the initiation of FAO reversal, the formation of cycle intermediates, and the synthesis of end products, the native regulatory regions of the fadE, fabI, and ydiO genes in BOX3.3 ∆4 derived strains were also replaced by the artificial regulatory element Ptrc-ideal-4-SDφ10.
All relevant recombinants formed profiles of the main secreted metabolites during anaerobic glucose utilization similar to those demonstrated by the parent strain BOX3.3 ∆4 (Table 3). The main glucose utilization products formed by the strains were acetic acid and ethanol, the yields of which were ~0.5 mol/mol and ~0.3 mol/mol, as well as lactic acid, the yield of which reached ~0.35 mol/mol.
The secretion by strains of a significant portion of consumed glucose in the form of acetic acid and ethanol, which are direct derivatives of acetyl-CoA, a key precursor in the reactions of reversed FAO, as well as lactic acid, which, along with ethanol, is a product of NADH-consuming reactions, indicated a low intensity of functioning in recombinants of the target biosynthetic pathway. However, strains BOX3.3 Δ4 Ptrc-id-4-fadE and BOX3.3 Δ4 Ptrc-id-4-fabI secreted significant amounts of butyric, caproic, and caprylic acids (Table 4), which are four-, six-, and eight-carbon products of a full-fledged one-, two- and three-turns FAO reversal. At the same time, the amount of butyric acid synthesized by the strains was considerably greater than the amount of six- and eight-carbon carboxylates formed by them. Apparently, this was due, first of all, to the insufficient specificity of acetyl-CoA-C-acetyltransferase AtoB to acyl-CoA substrates containing more than 4 carbon atoms in the hydrocarbon chain . Indeed, this enzyme is preferentially involved in the catalysis of the terminal stages of lipid degradation, while 3-ketoacyl-CoA thiolase FadA is involved in the cleavage of higher molecular weight intermediates .
The recombinant producers used in this study were model ones, while when constructing industrial strains, this problem can be solved by jointly enhancing the expression of the atoB and fadA genes in cells under the control of promoters with different strengths and regulation. Nevertheless, the obtained data allowed us to conclude that the use of FabI as acyl-CoA dehydrogenase promotes the multiple reversal of FAO to a greater extent than the use of FadE for this purpose. Indeed, the amount of caproic and caprylic acids synthesized by the BOX3.3 Δ4 Ptrc-id-4-fabI strain was 3.8 and 2.6 times higher than that of the BOX3.3 Δ4 Ptrc-id-4-fadE strain. However, nowadays, the enzymatic properties of FadE and FabI, as acyl-CoA dehydrogenases/enoyl-CoA reductases, have been poorly studied. Significantly different rates of specific acyl-CoA dehydrogenase activity for these proteins were reported, amounting to 0.019 µmol/mg/min and 0.001 µmol/mg/min for FadE and FabI against butyryl-CoA for strains expressing the corresponding genes in identical plasmids . At the same time, data on the Michaeles constant in relation to crotonyl-CoA obtained for the purified variant of recombinant FabI indicate its extremely low affinity for the corresponding substrate.
Thus, from the point of view of applied biotechnology, the achievement of FAO reversal should be based, in our opinion, on targeted manipulation of the expression levels of key genes with an analysis of the efficiency of substrate conversion into target products for the subsequent choice of a rational design strategy for industrial producers, and not on data on their enzymatic activity. At the same time, the specific activity of YdiO against butyryl-CoA was reported at 0.003 µmol/mg/min, which is comparable with the FabI properties . However, strain BOX3.3 Δ4 Ptrc-id-4-ydiO during anaerobic utilization of glucose did not synthesize noticeable amounts of marker compounds, which could indicate complete functional reversal of FAO (Table 4). Acyl-CoA dehydrogenase, which is involved in E. coli anaerobic FAO, is, by analogy with Clostridium, a complex enzymatic complex that involves flavoproteins in its functioning providing electron transport to the terminal acceptor. The corresponding proteins in E. coli encode the genes of the ydiQRST operon , therefore, to ensure the activity of the anaerobic acyl-CoA dehydrogenase YdiO, the expression of the genes of this operon was additionally increased in the BOX3.3 Δ4 Ptrc-id-4-ydiO strain. The final recombinant BOX3.3 Δ4 Ptrc-id-4-ydiO Ptrc-id-4-ydiQRST synthesized the full range of products of three-turn reversal of FAO (Table 4) with efficiency, however, decreased compared to strains BOX3.3 Δ4 Ptrc-id-4-fadE and BOX3.3 Δ4 Ptrc-id-4-fabI. In this regard, the need for the participation of other collateral enzymes in ensuring the maximum functional activity of the anaerobic acyl-CoA dehydrogenase YdiO/YdiQRST in E. coli, similar to that involved in energy conservation in the cells of obligate anaerobes and requiring the coordinated action of many enzymes involved in maintaining intracellular redox homeostasis could not be excluded . Taking into account the difficulty of ensuring the full functional activity of anaerobic acyl-CoA dehydrogenase for the reversal of FAO in recombinant cells, the data obtained indicated the preferential use of aerobic enzymes for the enforced inversion of this pathway to the biosynthetic direction.
As a result of the conducted studies, the ability of native acyl-CoA dehydrogenases to ensure multiple reversal of FAO in recombinant E. coli strains for potential production of industrially valuable compounds was characterized. It has been shown that the maximum efficiency of cycle turnover is achieved by increasing the level of expression of the gene encoding enoyl-ACP reductase/acyl-CoA dehydrogenase FabI in recombinant strains. An alternative could be an increase in the expression of the acyl-CoA dehydrogenase FadE gene, while maintaining the optimal balance between the activities of the enzymes involved in the target biosynthetic pathway.