Metabolic engineering of Yarrowia lipolytica for the production of isoprene

Abstract

Yarrowia lipolytica has recently emerged as a prominent microbial host for production of terpenoids. Its robust metabolism and growth in wide range of substrates offer several advantages at industrial scale. In the present study, we investigate the metabolic potential of Y. lipolytica to produce isoprene. Sustainable production of isoprene has been attempted through engineering several microbial hosts; however, the engineering studies performed so far are challenged with low titers. Engineering of Y. lipolytica, which have inherent high acetyl‐CoA flux could fuel precursors into the biosynthesis of isoprene and thus is an approach that would offer sustainable production opportunities. The present work, therefore, explores this opportunity wherein a codon‐optimized IspS gene (single copy) of Pueraria montana was integrated into the Y. lipolytica genome. With no detectable isoprene level during the growth or stationary phase of modified strain, attempts were made to overexpress enzymes from MVA pathway. GC‐FID analyses of gas collected during stationary phase revealed that engineered strains were able to produce detectable isoprene only after overexpressing HMGR (or tHMGR). The significant role of HMGR (tHMGR) in diverting the pathway flux toward DMAPP is thus highlighted in our study. Nevertheless, the final recombinant strains overexpressing HMGR (tHMGR) along with Erg13 and IDI showed isoprene titers of ~500 μg/L and yields of ~80 μg/g. Further characterization of the recombinant strains revealed high lipid and squalene content compared to the unmodified strain. Overall, the preliminary results of our laboratory‐scale studies represent Y. lipolytica as a promising host for fermentative production of isoprene.

Methanol bioconversion into C3, C4, and C5 platform chemicals by the yeast Ogataea polymorpha

“…In this study, two different variants of the isoprene synthase were tested, one from Pueraria montana (kudzu vine) and the other one from Populus alba (white poplar). Both of these genes have already been applied successfully for the heterologous production of isoprene in different microbial species [ 28 , 47 , 48 ]. As isoprene synthase genes typically have low turnover numbers and high Km values [ 49 ], it has been reported, that increasing the number of copies of isoprene synthase genes enhances the isoprene production [ 50 ].…”

Metabolic Engineering of Yarrowia lipolytica for Terpenoid Production: Tools and Strategies

“…Overexpression of HMG1, IDI, and ERG13 is a frequently adopted strategy to enhance the flux of the MVA pathway. 19,23 HMG1 in Y. lipolytica consists of 999 amino acid residues, of which the first 500 amino acid residues comprise several transmembrane structural domains and signal regulatory response elements, and the last 499 amino acid residues comprise the catalytic structural domain and NADPH binding region. 53 Studies have shown that N-terminal truncation of HMG1 can effectively increase the flux of the MVA pathway, preventing HMG1 from self-degradation mediated by its Nterminal structural domain, thereby stabilizing the spatial structure and enzymatic activity of HMG1 in the cyto-plasm.…”

“…10,12 Currently, overexpression of truncated HMG1 (tHMG1) has been widely used as a common means to enhance the availability of precursors in terpenoid-engineered strains. 29,44,52,66,68 However, some studies have shown that the N-terminal membrane structure of HMG1 plays a key but unknown role and that tHMG1 was not better than HMG1 in the synthesis of certain terpenoids, 19,38,45 and in a few cases Nterminal truncation even had a detrimental effect. 28,33,42,60 The case-specific effect of this enzyme requires the researchers to overexpress different types of HMG1 during the construction of different terpenoid-producing strains.…”

“…IDI plays a critical role in maintaining the balance between IPP and DMAPP. Overexpression of HMG1, IDI, and ERG13 is a frequently adopted strategy to enhance the flux of the MVA pathway. , …”

“…HMG1 in Y. lipolytica consists of 999 amino acid residues, of which the first 500 amino acid residues comprise several transmembrane structural domains and signal regulatory response elements, and the last 499 amino acid residues comprise the catalytic structural domain and NADPH binding region . Studies have shown that N-terminal truncation of HMG1 can effectively increase the flux of the MVA pathway, preventing HMG1 from self-degradation mediated by its N-terminal structural domain, thereby stabilizing the spatial structure and enzymatic activity of HMG1 in the cytoplasm. , Currently, overexpression of truncated HMG1 (tHMG1) has been widely used as a common means to enhance the availability of precursors in terpenoid-engineered strains. ,,,, However, some studies have shown that the N-terminal membrane structure of HMG1 plays a key but unknown role and that tHMG1 was not better than HMG1 in the synthesis of certain terpenoids, ,, and in a few cases N-terminal truncation even had a detrimental effect. ,,, The case-specific effect of this enzyme requires the researchers to overexpress different types of HMG1 during the construction of different terpenoid-producing strains. It is generally believed that NADH is usually more abundant than NADPH, , so introducing NADH-dependent exogenous HMG from Bordetella petrii or Ruegeria pomeroyi , or modifying the cofactor preference of the endogenous HMG might be a promising strategy to improve the MVA flux for terpenoids production.…”