Abstract
Microbial biocatalysis represents a promising alternative for the production of a variety of aromatic chemicals, where microorganisms are engineered to convert a renewable feedstock under mild production conditions into a valuable chemical building block. This study describes the rational engineering of the solvent-tolerant bacterium Pseudomonas taiwanensis VLB120 toward accumulation of L-phenylalanine and its conversion into the chemical building block t-cinnamate. We recently reported rational engineering of Pseudomonas toward L-tyrosine accumulation by the insertion of genetic modifications that allow both enhanced flux and prevent aromatics degradation. Building on this knowledge, three genes encoding for enzymes involved in the degradation of L-phenylalanine were deleted to allow accumulation of 2.6 mM of L-phenylalanine from 20 mM glucose. The amino acid was subsequently converted into the aromatic model compound t-cinnamate by the expression of a phenylalanine ammonia-lyase (PAL) from Arabidopsis thaliana. The engineered strains produced t-cinnamate with yields of 23 and 39% Cmol Cmol−1 from glucose and glycerol, respectively. Yields were improved up to 48% Cmol Cmol−1 from glycerol when two enzymes involved in the shikimate pathway were additionally overexpressed, however with negative impact on strain performance and reproducibility. Production titers were increased in fed-batch fermentations, in which 33.5 mM t-cinnamate were produced solely from glycerol, in a mineral medium without additional complex supplements. The aspect of product toxicity was targeted by the utilization of a streamlined, genome-reduced strain, which improves upon the already high tolerance of P. taiwanensis VLB120 toward t-cinnamate.
Highlights
Trans-cinnamate is an aromatic compound naturally occurring in plants, where it serves as central intermediate for the biosynthesis of a large number of substances, including coumarins, flavonoids, and phenylpropanoids (Chemler and Koffas, 2008; Vogt, 2010)
Microbes can be supplied with renewable feedstocks, and metabolic conversion enables the synthesis of diverse products under mild production conditions, either via native enzymes (Hosseinpour Tehrani et al, 2019) or by heterologous expression of foreign genes (Kallscheuer et al, 2019)
We recently reported a study on phenol production from L-tyrosine in P. taiwanensis VLB120, where 22 mutations in 50 different strains delivered a comprehensive insight on the rational engineering of L-tyrosine accumulation (Wynands et al, 2018)
Summary
Trans-cinnamate is an aromatic compound naturally occurring in plants, where it serves as central intermediate for the biosynthesis of a large number of substances, including coumarins, flavonoids, and phenylpropanoids (Chemler and Koffas, 2008; Vogt, 2010) It is widely used in industry for flavoring, pharmaceuticals, and cosmetics (Fausta et al, 1999; De et al, 2011; Vargas-Tah and Gosset, 2015) and can serve as precursor for bio-based drop-in chemicals such as styrene (McKenna and Nielsen, 2011) or for value-added chemicals such as the stilbene pinosylvin (van Summeren-Wesenhagen and Marienhagen, 2015). Commercial production currently happens from petroleum-based, non-renewable feedstocks in processes that demand high amounts of energy and release toxic by-products (Bruckner, 2010; Tietze et al, 2015) This comes along with increasing apprehension on global climate change and depleting aromatic fossil resources. From a biochemical engineering perspective, it is important to “begin with the end in mind” when developing microbial production strains (Straathof et al, 2019), and product toxicity is one important aspect for this
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