1 Introduction
Bio-manufacturing via microbial cell factories represents for an alternative production route for compounds previously obtained from natural resources or chemical synthesis [1,2], considering advantages in climate insensitivity, low land occupancy and environmental friendliness [3]. At present, the production of bulk chemicals, organic acids [4], fatty alcohols[5], and alkanes [6] have been realized in microbes. In addition, with the help of developed technologies of systems biology, protein engineering, cofactor engineering and computer-aided modeling [7,8], complex biosynthetic pathways have been designed to enable the productions of compounds like cannabidiol [9] and scopolamine [10]. In particular, the industrial-scale productions of artemisinin [11], farnesene [12] and squalene[13] demonstrate the greatest potential of microbial cell factories.
The auxotrophic marker gene is wildly used for convenient genetic manipulation during cell factory construction. However, the auxotrophic genotype might compromise the bio-production due to the insufficient supply of nutrients. For example, the auxotroph is often constructed by disrupting some essential genes for amino acids synthesis, such as leucine (LEU2 ), histidine (HIS3 ) and uracil (URA3 ) for convenient genetic manipulation in yeastSaccharomyces cerevisiae [14]. Traditional genetic manipulation relies on the insertion of heterologous metabolic pathways into the locus of auxotrophic markers [15]. Despite the fast development of molecular biology, auxotrophic selective markers are still used to maintain plasmid stability, and even sgRNA expression in the CRISPR/Cas9 system [16]. However, amino acids, as the precursors of protein synthesis, participate in various metabolic pathways and are crucial for microbial growth and production[17]. Therefore, the exogenous addition of amino acids during fermentations [15], may be not enough to support efficient cell growth to decrease product yields, and even misjudge the feasibility of metabolic engineering strategy[18], which, however, has not been paid much attentions by researchers.
Here, we evaluated the effects of eight commonly used auxotrophic markers, with grouping them into synthesis of acetyl-CoA, α-ketoglutarate, and succinyl-CoA derived modules (Figure 1), in cell growth and bio-productions of free fatty acid (FFA) in S. cerevisiae [19]. By comparing the prototrophic and auxotrophic strains with exogenous supplements of different concentrations of amino acids or nucleotides, we concluded that except for gene ADE2 , most auxotrophic strains possessed decreased cell growth and FFA production, which could be remedied by the higher concentrations of supplements. leu2 Δ damaged both growth and production even in 1000 mg/L of supplemented leucine, which is not suitable as a screening marker. This study reminded us to be cautious to use auxotrophic cell factory in bio-productions, and the recovery of these genes in fermentations is supposed to be essential for enhancing product yields.