Optimization of the IPP Precursor Supply for the Production of Lycopene, Decaprenoxanthin and Astaxanthin by Corynebacterium glutamicum

Abstract

The biotechnologically relevant bacterium Corynebacterium glutamicum, currently used for the million ton-scale production of amino acids for the food and feed industries, is pigmented due to synthesis of the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides. The precursors of carotenoid biosynthesis, isopenthenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate, are synthesized in this organism via the methylerythritol phosphate (MEP) or non-mevalonate pathway. Terminal pathway engineering in recombinant C. glutamicum permitted the production of various non-native C50 and C40 carotenoids. Here, the role of engineering isoprenoid precursor supply for lycopene production by C. glutamicum was characterized. Overexpression of dxs encoding the enzyme that catalyzes the first committed step of the MEP-pathway by chromosomal promoter exchange in a prophage-cured, genome-reduced C. glutamicum strain improved lycopene formation. Similarly, an increased IPP supply was achieved by chromosomal integration of two artificial operons comprising MEP pathway genes under the control of a constitutive promoter. Combined overexpression of dxs and the other six MEP pathways genes in C. glutamicum strain LYC3-MEP was not synergistic with respect to improving lycopene accumulation. Based on C. glutamicum strain LYC3-MEP, astaxanthin could be produced in the milligrams per gram cell dry weight range when the endogenous genes crtE, crtB, and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were coexpressed with the genes for lycopene cyclase and β-carotene hydroxylase from Pantoea ananatis and carotene C(4) oxygenase from Brevundimonas aurantiaca.

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-β- d -Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum

“…In the newly constructed (ϩ)-valencene producer strain VLC4, the CnVS-encoding gene was expressed after its adaptation to the codon usage of C. glutamicum. In strain VLC6, the genes coding for IspA and CnVS were combined on a single vector, allowing the introduction of a second IPTG-inducible vector for overexpression of the genes encoding the 1-deoxy-D-xylulose 5-phosphate synthase (dxs) and isopentenyl pyrophosphate isomerase (idi) to enhance the supply of the precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) (18), and consequently, of FPP. Since CnVS gene expression perturbed growth, we presumed that the optimization and timing of CnVS expression would be one key aspect to elevate (ϩ)-valencene production (Fig.…”

“…orynebacterium glutamicum represents one of the most important biotechnological platform organisms and massively contributes to the industrial production of amino acids (1)(2)(3)(4)(5), but it has also been engineered, for instance, for the production of lower alcohols (6)(7)(8)(9), organic acids (10-13), diamines (14,15), and carotenoids (16)(17)(18). However, currently applied expression setups show only moderate performance regarding both precise control and expression homogeneity.…”

Engineering of Corynebacterium glutamicum for growth and production of L-ornithine, L-lysine, and lycopene from hexuronic acids

“…It is often observed that too high levels of a transmembrane protein such as a transport protein results in growth perturbation [53,54]. In addition, expression levels of several genes of a pathway may need to be tuned to avoid accumulation of potentially inhibitory intermediates as demonstrated for C. glutamicum engineered for decaprenoxanthin overproduction [23]. It is not known if the recombinant C. glutamicum strain WT(pHexA)(pVWEX1-exuT) would be able to grow with sugar acids related to the aldohexuronates Dgalacturonate and D-glucuronate since ExuT is specific for aldohexuronate uptake.…”

“…Other strains include C. glutamicum ORN1 [14], C. glutamicum DM1933 [37], and C. glutamicum ΔcrtYEB [23] that are derived from the wild-type strain ( Table 1). The hexuronic acid utilization and transporter genes originated from E. coli MG1655, whereas the strain E. coli DH5α [38] was used for plasmid construction.…”

“…From its initial use as a natural L-glutamate producer [10], it is currently used for production of other amino acids such as L-lysine, L-ornithine, L-methionine, and Laspartate [11][12][13][14]. However, its importance has further increased as it was for production of of non-natural products [9] such as isobutanol [15], ethanol [16], putrescine [14,17,18], cadaverine [19], carotenoids and terpenoids [20][21][22][23][24][25], and xylitol [26].…”