Targeting metabolic driving and intermediate influx in lysine catabolism for high-level glutarate production

Abstract

Various biosynthetic pathways have been designed to explore sustainable production of glutarate, an attractive C5 building block of polyesters and polyamides. However, its efficient production has not been achieved in Escherichia coli . Here, we use E. coli native lysine catabolic machinery for glutarate biosynthesis. This endogenous genes-only design can generate strong metabolic driving force to maximize carbon flux toward glutarate biosynthesis by replenishing glutamate and NAD(P)H for lysine biosynthesis, releasing lysine feedback inhibition, and boosting oxaloacetate supply. We use native transporters to overcome extracellular accumulation of cadaverine and 5-aminovalerate. With these efforts, both high titer (54.5 g L −1 ) and high yield (0.54 mol mol −1 glucose) of glutarate production are achieved under fed-batch conditions. This work demonstrates the power of redirecting carbon flux and the role of transporters to decrease intermediate accumulation.

Bacterial synthesis of C3-C5 diols via extending amino acid catabolism

“…To further improve the platform performance, debottlenecking the upstream AA-to-ω-HA conversion by screening superior enzymes would be most pivotal, considering the high performance of the Carcatalyzed downstream ω-HA-to-diol conversion. Second, increasing the AA import or reducing the intermediate export via transporter systems would maximize AA utilization and thus the final product yield, as extensively exemplified in previous work (58,62,63). Finally, it is expected that additional genetic manipulations to enhancing AA flux in microbial host, or switching to the other well-developed AA hyperproducers like C. glutamicum, will further improve diol production.…”

“…6). Finally, to increase the supply of lysine pathway precursor OAA, we knocked out iclR, which encodes a repressor regulator of the glyoxylate bypass that serves as an alternative mechanism of replenishing oxaloacetate (21,58). The final 1,5-PDO titer produced by strain 5PDO-7 with iclR deletion reached 0.97 g l −1 at 48 h with a yield of 0.08 mol mol −1 glucose (11.2% of the theoretical maximum).…”

Transporter engineering in microbial cell factories: the ins, the outs, and the in-betweens

“…When E. coli was used for the production of glutarate through the native lysine pathway, biosynthetic intermediates 5-aminovalerate and cadaverine were secreted at 1.19 g/L and 1.66 g/L, respectively. Overexpression of uptake transporters GabP and PotE improved the re-uptake of these intermediates and enhanced glutarate production by 72% and 11%, respectively [ 4 • ]. In another example, E. coli was used to produce ω-hydroxy palmitic acid from glucose, but at the same time, palmitic acid was secreted at ∼100 mg/L.…”

“…The most straightforward method is to search for homologs of known transporters to find transporters for similar substrates [ 33 •• ] or transporters with improved kinetics [ 2 • ]. In bacteria, transporters can sometimes be found in their biosynthetic gene clusters, as is the case for the minimycin transporter MinT of Streptomyces hygroscopicus JCM 712 [ 34 ] and gabP in the gabDTP operon of E. coli [ 4 • ] ( Figure 2 a). AntiSMASH is a powerful tool to identify gene clusters, and it also annotates transporters within the gene clusters [ 35 ].…”

“…In the case of multi-step biosynthetic pathways, a common problem is the leakage of the biosynthetic intermediates from the cell. If the leakage of intermediates can be prevented by manipulating transporter activity ( Figure 1 c), then more carbon will be channeled into the final product, improving the process economics [ 4 • ].

Figure 1 Illustrative case stories of transporter engineering; (a) The alternative substrate d -galacturonate is taken up and converted into meso -galactaric acid while glucose is used for biomass production; (b) The Gal2 transporter is coupled to xylose isomerase, so that when xylose is taken up, it is channeled to the xylose isomerase, decreasing by-product formation and increasing ethanol production; (c) The leaked intermediates cadavarine and 5-aminovalerate are re-imported from the medium into the cell by PotE and GabP, respectively, to increase the production of the pathway product glutarate; (d) The pathway product lysine is exported by YbjE to reduce feedback inhibition; (e) FATP1 exports the fatty alcohol products, reducing toxicity and improving growth; (f) Overexpression of acrE , mdtC , and mdtE and deletion of cmr simultaneously improves titer limitations by exporting the fatty acid products out of the cell.

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