Production of 4-Hydroxybenzoic Acid by an Aerobic Growth-Arrested Bioprocess Using Metabolically Engineered Corynebacterium glutamicum

Abstract

was metabolically engineered to produce 4-hydroxybenzoic acid (4-HBA), a valuable aromatic compound used as a raw material for the production of liquid crystal polymers and paraben. was found to have a higher tolerance to 4-HBA toxicity than previously reported hosts used for the production of genetically engineered 4-HBA. To obtain higher titers of 4-HBA, we employed a stepwise overexpression of all seven target genes in the shikimate pathway in Specifically, multiple chromosomal integrations of a mutated gene from, encoding a 3-deoxy-d-arabinoheptulosonic acid 7-phosphate (DAHP) synthase, and wild-type from, encoding chorismate synthase, shikimate kinase, and 3-dehydroquinate synthase, were effective in increasing product titers. The last step of the 4-HBA biosynthesis pathway was recreated in by expressing a highly 4-HBA-resistant chorismate pyruvate-lyase (UbiC) from the intestinal bacterium To enhance the yield of 4-HBA, we reduced the formation of by-products, such as 1,3-dihydroxyacetone and pyruvate, by deleting , a gene coding for a haloacid dehalogenase superfamily phosphatase, and, a gene coding for a pyruvate kinase, from the bacterial chromosome. The maximum concentration of 4-HBA produced by the resultant strain was 36.6 g/liter, with a yield of 41% (mol/mol) glucose after incubation for 24 h in minimal medium in an aerobic growth-arrested bioprocess using a jar fermentor. To our knowledge, this is the highest concentration of 4-HBA produced by a metabolically engineered microorganism ever reported. Since aromatic compound 4-HBA has been chemically produced from petroleum-derived phenol for a long time, eco-friendly bioproduction of 4-HBA from biomass resources is desired in order to address environmental issues. In microbial chemical production, product toxicity often causes problems, but we confirmed that wild-type has high tolerance to the target 4-HBA. A growth-arrested bioprocess using this microorganism has been successfully used for the production of various compounds, such as biofuels, organic acids, and amino acids. However, no production method has been applied for aromatic compounds to date. In this study, we screened for a novel final reaction enzyme possessing characteristics superior to those in previously employed microbial 4-HBA production. We demonstrated that the use of the highly 4-HBA-resistant UbiC from the intestinal bacterium is very effective in increasing 4-HBA production.

Microbial production of methyl anthranilate, a grape flavor compound

“…2B), and pgi, zwf, tkt, opcA, pgl, and tal known to enhance flux through the pentose phosphate pathway (SI Appendix, Fig. S13), which were target genes similarly manipulated for the overproduction of shikimic acid, 4-hydroxybenzoic acid, L-ornithine, and L-arginine in C. glutamicum (22)(23)(24)(25). To this end, the following strains were constructed: YTM3 (with the native promoters of aroK and aroB replaced with the strong constitutive sod promoter in YTM2 strain), YTM4 (with the start codon of pgi changed from ATG to GTG for down-regulation and of zwf changed from GTG to ATG for up-regulation in YTM3 strain), YTM5 (with the native promoter of tkt replaced with sod promoter in YTM4 strain), as well as plasmid pEKG (overexpressing aroG S180F encoding a feedback-resistant DAHP synthase).…”

“…Glycerol formation was also observed in the fermentation broth, and thus the hdpA gene involved in glycerol formation pathway was also deleted (SI Appendix, Fig. S20) (22,23) in YTM2 strain, generating YTM8. As a result, two-phase fed-batch culture of YTM8 strain harboring pSH36HTc and pEKGH produced MANT to a 9.3% higher concentration of 5.74 g/L at 110 h (Figs.…”

Bioprocess Optimization for the Production of Aromatic Compounds With Metabolically Engineered Hosts: Recent Developments and Future Challenges

“…This strain was then used and allowed a PHBA formation from CHO, with a titer of 2.9 g L −1 and yield of 3.1 mg g glucose −1 , in a fed-batch process (Averesch et al, 2017). To date, the highest PHBA titer was reported by Kitade et al (2018) in C. glutamicum (Table 1). This was achieved by chromosomal integration of aroG from E. coli and wild-type aroCKB from C. glutamicum, encoding chorismate synthase, shikimate kinase, and 3-dehydroquinate synthase.…”

Chemistry of Secondary Metabolites

“…Therefore, biotransformation of phenolic acids gives a monetarily reasonable and supportable methods for delivering helpful materials for society [53]. P-hydroxybenzoic corrosive utilized as a crude material for the creation of fluid gem polymers and paraben [54]. Caffeic Corrosive Phenethyl Ester (CAPE) goes about as a particular inhibitor of NF-κB in bosom malignancy cells [55].…”