Unraveling the specific regulation of the shikimate pathway for tyrosine accumulation in Bacillus licheniformis

Abstract

l-Tyrosine serves as a common precursor for multiple valuable secondary metabolites. Synthesis of this aromatic amino acid in Bacillus licheniformis occurs via the shikimate pathway, but the underlying mechanisms involving metabolic regulation remain unclear. In this work, improved l-tyrosine accumulation was achieved in B. licheniformis via co-overexpression of aroGfbr and tyrAfbr from Escherichia coli to yield strain 45A12, and the l-tyrosine titer increased to 1005 mg/L with controlled glucose feeding. Quantitative RT-PCR results indicated that aroA, encoding DAHP synthase, and aroK, encoding shikimate kinase, were feedback-repressed by the end product l-tyrosine in the modified strain. Therefore, the native aroK was first expressed with multiple copies to yield strain 45A13, which could accumulate 1201 mg/L l-tyrosine. Compared with strain 45A12, the expression of aroB and aroF in strain 45A13 was upregulated by 21% and 27%, respectively, which may also have resulted in the improvement of l-tyrosine production. Furthermore, supplementation with 5 g/L shikimate enhanced the l-tyrosine titers of 45A12 and 45A13 by 29.1% and 24.0%, respectively. However, the yield of l-tyrosine per unit of shikimate decreased from 0.365 to 0.198 mol/mol after aroK overexpression in strain 45A12, which suggested that the gene product was also involved in uncharacterized pathways. This study provides a good starting point for further modification to achieve industrial-scale production of l-tyrosine using B. licheniformis, a generally recognized as safe workhorse.

Combining metabolite doping and metabolic engineering to improve 2-phenylethanol production by engineered cyanobacteria

“…Nowadays, the co-overexpression of both feedback-inhibition resistant E. coli enzymes is common to gain aromatics production, mostly in E. coli and yeast ( Báez-Viveros et al, 2004 ; Zhang et al, 2014a ; Rodriguez et al, 2014 , 2015 ; Wang et al, 2019 ; Koma et al, 2020 ), as well as in cyanobacteria ( Ni et al, 2018 ; Brey et al, 2020 ; Deshpande, Vue and Morgan, 2020 ). Additionally, in the literature another reaction has been reported as high flux controlling step ( Luttik et al, 2008 ; Takai A, Nishi R, Joe Y, 2009; Darmawi et al, 2012 ; Rodriguez et al, 2015 ; Xu et al, 2019 ), i.e. shikimate kinase-mediated reaction, the conversion of shikimate and ATP in shikimate-3-phosphate and ADP.…”

“…E. coli expresses three genes encoding DAHP synthases, which are respectively sensitive to a specific AAA (Jossek, Bongaerts and Sprenger, 2001;Chávez-Béjar et al, 2012;Noda and Kondo, 2017). Furthermore, as already reported in some bacteria and yeast, the shikimate kinase (EC 2.7.1.71), which catalyzes the conversion of shikimate in shikimate-3-phosphate (the fifth step in SKP), can relevantly affect the carbon flow by acting as bottleneck in the SKP (Luttik et al, 2008;Takai et al, 2009;Xu et al, 2019). So far, numerous efforts have been already pursued to yield aromatic chemicals by genetically and non-genetically modified microorganisms.…”

Transcriptional Changes in the Xylose Operon in Bacillus licheniformis and Their Use in Fermentation Optimization

“…The RT-qPCR amplification efficiencies were calculated using the formula E=false(10−1/s−1false)×100%, where s is the slope of the standard curve with several dilutions of cDNA [49]. The expression of the xylA gene was determined relative to that of rpsE [50,51] by RT-qPCR.…”

Differences in Gluco and Galacto Substrate-Binding Interactions in a Dual 6Pβ-Glucosidase/6Pβ-Galactosidase Glycoside Hydrolase 1 Enzyme from Bacillus licheniformis

“…The Gram-positive bacterium B. licheniformis is favorable to industrial purposes, having characteristics such as easy genetic manipulation, agreeable to industrial fermentation conditions, generally recognized as safe (GRAS) status, and possessing probiotic attributes. − In this study, a GH1 enzyme from B. licheniformis ( Bl BglC) was analyzed to confirm its enzyme activity and to study its substrate specificity.…”