Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities

Abstract

Aliphatic hydrocarbons such as fatty alcohols and petroleum-derived alkanes have numerous applications in the chemical industry. In recent years, the renewable synthesis of aliphatic hydrocarbons has been made possible by engineering microbes to overaccumulate fatty acids. However, to generate end products with the desired physicochemical properties (e.g., fatty aldehydes, alkanes, and alcohols), further conversion of the fatty acid is necessary. A carboxylic acid reductase (CAR) from Mycobacterium marinum was found to convert a wide range of aliphatic fatty acids (C 6 –C 18 ) into corresponding aldehydes. Together with the broad-substrate specificity of an aldehyde reductase or an aldehyde decarbonylase, the catalytic conversion of fatty acids to fatty alcohols (C 8 –C 16 ) or fatty alkanes (C 7 –C 15 ) was reconstituted in vitro. This concept was applied in vivo , in combination with a chain-length-specific thioesterase, to engineer Escherichia coli BL21(DE3) strains that were capable of synthesizing fatty alcohols and alkanes. A fatty alcohol titer exceeding 350 mg·L −1 was obtained in minimal media supplemented with glucose. Moreover, by combining the CAR-dependent pathway with an exogenous fatty acid-generating lipase, natural oils (coconut oil, palm oil, and algal oil bodies) were enzymatically converted into fatty alcohols across a broad chain-length range (C 8 –C 18 ). Together with complementing enzymes, the broad substrate specificity and kinetic characteristics of CAR opens the road for direct and tailored enzyme-catalyzed conversion of lipids into user-ready chemical commodities.

Selective Enzymatic Transformation to Aldehydes in vivo by Fungal Carboxylate Reductase from Neurospora crassa

“…With the aim to identify the primary sequence of Neurospora crassa CAR, four literature known CAR enzyme sequences ( Ni CAR:5 Q6RKB1.1, Mm CAR:6 B2HN69, Sr CAR:8 WP_013138593.1 and At CAR:7 XP_001212808.1) were used as templates for a blastp search against the Gen‐Bank non‐redundant protein sequences of Neurospora crassa (taxid:5241). The hit with highest scores was a hypothetical protein with NCBI accession code XP_955820.1.…”

“…Carboxylate reductase (CAR) enzymes exhibit a broad substrate tolerance for the conversion of organic acids to the respective aldehydes 4. However, only few CAR enzyme sequences have been elucidated5, 6, 7, 8, 9, 10, 11, 12 and are available for biocatalysis to date, although Nature provides a great versatility of organisms with the capability to catalyze this reaction. [4,13–18] The reduction of salicylic acid,19 benzoic acid and derivatives20 as well as cinnamic acid and derivatives21 has been observed in the ascomycete Neurospora crassa ( Nc ).…”

Organic reactions for the electrochemical and photochemical production of chemical fuels from CO2 – The reduction chemistry of carboxylic acids and derivatives as bent CO2 surrogates

“…The reduction of carboxylic acids to aldehydes can be performed enzymatically by carboxylic acid reductase(CAR) [79,80]. The enzyme is able to convert non-activated carboxylates to the corresponding reduced aldehydes without overreduction to the corresponding alcohols [79,80].…”

“…The enzyme is able to convert non-activated carboxylates to the corresponding reduced aldehydes without overreduction to the corresponding alcohols [79,80].…”

“…In conjunction with an additional reducing enzyme, the intermediate aldehyde can be further reduced to an alkane [79,80].…”