Fernando Pérez-García

Improved fermentative production of gamma-aminobutyric acid via the putrescine route: systems metabolic engineering for production from glucose, amino sugars and xylose.

Gamma‐aminobutyric acid (GABA) is a non‐protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2‐pyrrolidone can be chemically converted to the biodegradable plastic polyamide‐4. In metabolism, GABA can be synthesized either by decarboxylation of l‐glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine‐based GABA production was characterized by accumulation of by‐products such as N‐acetyl‐putrescine. Their formation was abolished by deletion of the spermi(di)ne N‐acetyl‐transferase gene snaA. To improve provision of l‐glutamate as precursor 2‐oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2‐oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory form by changing amino acid residue 15 from threonine to alanine. Putrescine‐based GABA production by the strains described here led to GABA titers up to 63.2 g L−1 in fed‐batch cultivation at maximum volumetric productivities up to 1.34 g L−1 h−1, the highest volumetric productivity for fermentative GABA production reported to date. Moreover, GABA production from the carbon sources xylose, glucosamine, and N‐acetyl‐glucosamine that do not have competing uses in the food or feed industries was established. Biotechnol. Bioeng. 2017;114: 862–873.

A new metabolic route for the fermentative production of 5-aminovalerate from glucose and alternative carbon sources

Here, a new metabolic pathway for the production of 5-aminovalerate (5AVA) from l-lysine via cadaverine as intermediate was established and this three-step-pathway comprises l-lysine decarboxylase (LdcC), putrescine transaminase (PatA) and γ-aminobutyraldehyde dehydrogenase (PatD). Since Corynebacterium glutamicum is used for industrial l-lysine production, the pathway was established in this bacterium. Upon expression of ldcC, patA and patD from Escherichia coli in C. glutamicum wild type, production 5AVA was achieved. Enzyme assays revealed that PatA and PatD also converted cadaverine to 5AVA. Eliminating the by-products cadaverine, N-acetylcadaverine and glutarate in a genome-streamlined l-lysine producing strain expressing ldcC, patA and patD improved 5AVA production to a titer of 5.1gL-1, a yield of 0.13gg-1 and a volumetric productivity of 0.12gL-1h-1. Moreover, 5AVA production from the alternative feedstocks starch, glucosamine, xylose and arabinose was established.

Fermentative production of L-pipecolic acid from glucose and alternative carbon sources.

Corynebacterium glutamicum is used for the million-ton scale production of amino acids and has recently been engineered for production of the cyclic non-proteinogenic amino acid L-pipecolic acid (L-PA). In this synthetic pathway L-lysine was converted to L-PA by oxidative deamination, dehydration and reduction by L-lysine 6-dehydrogenase (deaminating) from Silicibacter pomeroyi and pyrroline 5-carboxylate reductase from C. glutamicum. However, production of L-PA occurred as by-product of L-lysine production only. Here, the author show that abolishing L-lysine export by the respective gene deletion resulted in production of L-PA as major product without concomitant lysine production while the specific growth rate was reduced due to accumulation of high intracellular lysine concentrations. Increasing expression of the genes encoding L-lysine 6-dehydrogenase and pyrroline 5-carboxylate reductase in C. glutamicum strain PIPE4 increased the L-PA titer to 3.9 g L-1 , and allowed faster growth and, thus, a higher volumetric productivity of 0.08 ± 0.00 g L-1 h-1 respectively. Secondly, expression of heterologous genes for utilization of glycerol, xylose, glucosamine, and starch in strain PIPE4 enabled L-PA production from these alternative carbon sources. Third, in a glucose/sucrose-based fed-batch fermentation with C. glutamicum PIPE4 L-PA was produced to a titer of 14.4 g L-1 with a volumetric productivity of 0.21 g L-1 h-1 and an overall yield of 0.20 g g-1 .

Coproduction of cell-bound and secreted value-added compounds: simultaneous production of carotenoids and amino acids by Corynebacterium glutamicum

Corynebacterium glutamicum is used for production of the food and feed amino acids l-glutamate and l-lysine at the million-ton-scale. One feed formulation of l-lysine simply involves spray-drying of the fermentation broth, thus, including secreted l-lysine and C. glutamicum cells which are pigmented by the C50 carotenoid decaprenoxanthin. C. glutamicum has been engineered for overproduction of various compounds including carotenoids. In this study, C. glutamicum was engineered for coproduction of a secreted amino acid with a cell-bound carotenoid. Asa proof of principle, coproduction of l-glutamate with the industrially relevant astaxanthin was shown. This strategy was applied to engineer l-lysine overproducing strains for combined overproduction of secreted l-lysine with the cell-bound carotenoids decaprenoxanthin, lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin. By fed-batch fermentation 48g/Ll-lysine and 10mg/L astaxanthin were coproduced. Moreover, C. glutamicum was engineered for coproduction of l-lysine and β-carotene from xylose and arabinose as alternative feedstocks.

Biotechnological production of mono- and diamines using bacteria: recent progress, applications and perspectives

Common plastics such as polyamides are derived typically from petroleum or natural gas. Fossil-based polyamide production often involves toxic precursors or intermediates. By contrast, bio-based polyamides offer a realistic alternative. Bio-based routes to monomeric precursors of polyamides such as diamines, dicarboxylic acids, and omega-amino acids have been developed. Recent advances in the metabolic engineering of the biotechnologically relevant Escherichia coli and Corynebacterium glutamicum for the production of monoalkylamines such as omega-amino acids as well as diamines are presented.

Biotypes analysis of Corynebacterium glutamicum growing in dicarboxylic acids demonstrates the existence of industrially-relevant intra-species variations.

Production enhancement of industrial microbial products or strains has been traditionally tackled by mutagenesis with chemical methods, irradiation or genetic manipulation. However, the final yield increase must go hand in hand with the resistance increasing against the usual inherent toxicity of the final products. Few studies have been carried out on resistance improvement and even fewer on the initial selection of naturally-generated biotypes, which could decrease the artificial mutagenesis. This fact is vital in the case of GRAS microorganisms as Corynebacterium glutamicum involved in food, feed and cosmetics production. The characteristic wide diversity and plasticity in terms of their genetic material of Actinobacteria eases the biotypes generation. Thus, differences in morphology, glutamate and lysine production and growth in media supplemented with dicarboxylic acids were analysed in four biotypes of C. glutamicum ATCC 13032. A 2D-DIGE analysis of these biotypes growing with itaconic acid allowed us to define their differences. Thus, an optimized central metabolism and better protection against the generated stress conditions present the CgL biotype as a suitable platform for production of itaconic acid, which is used as a building block (e.g.: acrylic plastic). This analysis highlights the preliminary biotypes screening as a way to reach optimal industrial productions.

Transport and metabolic engineering of the cell factory Corynebacterium glutamicum

Corynebacterium glutamicum has a long and successful history in the biotechnological production of the amino acids l-glutamate and l-lysine. In the recent years, C. glutamicum has been engineered for the production of a broad catalog of value-added compounds including organic acids, vitamins, terpenoids and proteins. Moreover, this bacterium has been engineered to realize a flexible carbon source concept enabling product formation from various second generation feedstocks without competing uses in human and animal nutrition. In this review, we highlight transport engineering to improve product export and substrate uptake or to avoid loss of intermediates by excretion as well as the application of new metabolic engineering concepts for C. glutamicum strain development including the use of designed synthetic Escherichiacoli-C. glutamicum consortia. As examples, pathway extension of l-lysine and l-glutamate biosynthesis to produce derived value-added chemicals is described. The described examples of C. glutamicum strain engineering reflect strategies to cope with the increasing complexity of biotechnological processes that are required for successful applications in the bioeconomy.