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.