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Production of Biopolyamide Precursors 5-Amino Valeric Acid and Putrescine From Rice Straw Hydrolysate by Engineered Corynebacterium glutamicum.

ABSTRACT: The non-proteinogenic amino acid 5-amino valeric acid (5-AVA) and the diamine putrescine are potential building blocks in the bio-polyamide industry. The production of 5-AVA and putrescine using engineered Corynebacterium glutamicum by the co-consumption of biomass-derived sugars is an attractive strategy and an alternative to their petrochemical synthesis. In our previous work, 5-AVA production from pure xylose by C. glutamicum was shown by heterologously expressing xylA from Xanthomonas campestris and xylB from C. glutamicum. Apart from this AVA Xyl culture, the heterologous expression of xylA _Xc and xylB _Cg was also carried out in a putrescine producing C. glutamicum to engineer a PUT Xyl strain. Even though, the pure glucose (40 g L^-1) gave the maximum product yield by both the strains, the utilization of varying combinations of pure xylose and glucose by AVA Xyl and PUT Xyl in CGXII synthetic medium was initially validated. A blend of 25 g L^-1 of glucose and 15 g L^-1 of xylose in CGXII medium yielded 109 ± 2 mg L^-1 putrescine and 874 ± 1 mg L^-1 5-AVA after 72 h of fermentation. Subsequently, to demonstrate the utilization of biomass-derived sugars, the alkali (NaOH) pretreated-enzyme hydrolyzed rice straw containing a mixture of glucose (23.7 g L^-1) and xylose (13.6 g L^-1) was fermented by PUT Xyl and AVA Xyl to yield 91 ± 3 mg L^-1 putrescine and 260 ± 2 mg L^-1 5-AVA, respectively, after 72 h of fermentation. To the best of our knowledge, this is the first proof of concept report on the production of 5-AVA and putrescine using rice straw hydrolysate (RSH) as the raw material.

SUBMITTER: Sasikumar K

PROVIDER: S-EPMC8044859 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background:Succinate has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. However, efficient methods for the production of succinate from lignocellulosic feedstock were rarely reported. Nevertheless, Corynebacterium glutamicum was engineered to efficiently produce succinate from glucose in our previous study. Results:In this work, C. glutamicum was engineered for efficient succinate production from lignocellulosic hydrolysate. First, xylose utilization of C. glutamicum was optimized by heterologous expression of xylA and xylB genes from different sources. Next, xylA and xylB from Xanthomonas campestris were selected among four candidates to accelerate xylose consumption and cell growth. Subsequently, the optimal xylA and xylB were co-expressed in C. glutamicum strain SAZ3 (?ldhA?pta?pqo?catPsod-ppcPsod-pyc) along with genes encoding pyruvate carboxylase, citrate synthase, and a succinate exporter to achieve succinate production from xylose in a two-stage fermentation process. Xylose utilization and succinate production were further improved by overexpressing the endogenous tkt and tal genes and introducing araE from Bacillus subtilis. The final strain C. glutamicum CGS5 showed an excellent ability to produce succinate in two-stage fermentations by co-utilizing a glucose-xylose mixture under anaerobic conditions. A succinate titer of 98.6 g L-1 was produced from corn stalk hydrolysate with a yield of 0.87 g/g total substrates and a productivity of 4.29 g L-1 h-1 during the anaerobic stage. Conclusion:This work introduces an efficient process for the bioconversion of biomass into succinate using a thoroughly engineered strain of C. glutamicum. To the best of our knowledge, this is the highest titer of succinate produced from non-food lignocellulosic feedstock, which highlights that the biosafety level 1 microorganism C. glutamicum is a promising platform for the envisioned lignocellulosic biorefinery.

Project description:?-D-Glucosylglycerol (?GG) has beneficial functions as a moisturizing agent in cosmetics and potential as a health food material, and therapeutic agent. ?GG serves as compatible solute in various halotolerant cyanobacteria such as Synechocystis sp. PCC 6803, which synthesizes ?GG in a two-step reaction: The enzymatic condensation of ADP-glucose and glycerol 3-phosphate by GG-phosphate synthase (GGPS) is followed by the dephosphorylation of the intermediate by the GG-phosphate phosphatase (GGPP). The Gram-positive Corynebacterium glutamicum, an industrial workhorse for amino acid production, does not utilize ?GG as a substrate and was therefore chosen for the development of a heterologous microbial production platform for ?GG.Plasmid-bound expression of ggpS and ggpP from Synechocystis sp. PCC 6803 enabled ?GG synthesis exclusively in osmotically stressed cells of C. glutamicum (pEKEx2-ggpSP), which is probably due to the unique intrinsic control mechanism of GGPS activity in response to intracellular ion concentrations. C. glutamicum was then engineered to optimize precursor supply for ?GG production: The precursor for ?GG synthesis ADP-glucose gets metabolized by both the glgA encoded glycogen synthase and the otsA encoded trehalose-6-phosphate synthase. Upon deletion of both genes the ?GG concentration in culture supernatants was increased from 0.5 mM in C. glutamicum (pEKEx3-ggpSP) to 2.9 mM in C. glutamicum ?otsA IMglgA (pEKEx3-ggpSP). Upon nitrogen limitation, which inhibits synthesis of amino acids as compatible solutes, C. glutamicum ?otsA IMglgA (pEKEx3-ggpSP) produced more than 10 mM ?GG (about 2 g L-1).Corynebacterium glutamicum can be engineered as efficient platform for the production of the compatible solute ?GG. Redirection of carbon flux towards ?GG synthesis by elimination of the competing pathways for glycogen and trehalose synthesis as well as optimization of nitrogen supply is an efficient strategy to further optimize production of ?GG.

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Production of Biopolyamide Precursors 5-Amino Valeric Acid and Putrescine From Rice Straw Hydrolysate by Engineered Corynebacterium glutamicum.

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